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  Fluorescence Resonance Energy Transfer Detection of Synaptophysin I and Vesicle-Associated Membrane Protein 2 Interactions During Exocytosis From Single Live …
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  See discussions, stats, and author profiles for this publication at: http://www.researchgate.net/publication/11206712 Fluorescence Resonance Energy TransferDetection of Synaptophysin I and Vesicle-associated Membrane Protein 2...  Article   in  Molecular Biology of the Cell · September 2002 DOI: 10.1091/mbc.E02-01-0036 · Source: PubMed CITATIONS 50 READS 33 5 authors , including: Some of the authors of this publication are also working on these related projects: Post-translational modifications in neurodegenerative diseases   View projectMaria PennutoUniversità degli Studi di Trento 68   PUBLICATIONS   1,419   CITATIONS   SEE PROFILE David D DunlapEmory University 111   PUBLICATIONS   1,662   CITATIONS   SEE PROFILE Andrea ContestabileIstituto Italiano di Tecnologia 32   PUBLICATIONS   869   CITATIONS   SEE PROFILE Flavia ValtortaOspedale di San Raffaele Istituto di Ricovero… 173   PUBLICATIONS   8,037   CITATIONS   SEE PROFILE All content following this page was uploaded by Maria Pennuto on 02 January 2017. The user has requested enhancement of the downloaded file. All in-text references underlined in blue are added to the srcinal documentand are linked to publications on ResearchGate, letting you access and read them immediately.  Molecular Biology of the CellVol. 13, 2706–2717, August 2002 Fluorescence Resonance Energy Transfer Detection ofSynaptophysin I and Vesicle-associated MembraneProtein 2 Interactions during Exocytosis from SingleLive Synapses Maria Pennuto,* David Dunlap,* Andrea Contestabile,* Fabio Benfenati, † and Flavia Valtorta* ‡ *Department of Neuroscience, S. Raffaele Scientific Institute and “Vita-Salute” University, Milan, Italy;and  † Department of Experimental Medicine, Section of Human Physiology, University of Genoa, Italy Submitted January 22, 2002; Revised March 28, 2002; Accepted May 7, 2002Monitoring Editor: Hugh R.B. Pellham To investigate the molecular interactions of synaptophysin I and vesicle-associated membraneprotein 2 (VAMP2)/synaptobrevin II during exocytosis, we have used time-lapse videomicros-copy to measure fluorescence resonance energy transfer in live neurons. For this purpose,fluorescent protein variants fused to synaptophysin I or VAMP2 were expressed in rat hippocam-pal neurons. We show that synaptophysin I and VAMP2 form both homo- and hetero-oligomerson the synaptic vesicle membrane. When exocytosis is stimulated with   -latrotoxin, VAMP2dissociates from synaptophysin I even in the absence of appreciable exocytosis, whereas synap-tophysin I oligomers disassemble only upon incorporation of the vesicle with the plasma mem- brane. We propose that synaptophysin I has multiple roles in neurotransmitter release, regulatingVAMP2 availability for the soluble  N  -ethylmaleimide-sensitive factor attachment protein receptorcomplex and possibly participating in the late steps of exocytosis. INTRODUCTION Neurotransmitter release comprises a series of steps involv-ing synaptic vesicle (SV), plasma membrane, and cytosolicproteins. The molecular characterization of the repertoire of proteins involved has been the goal of a large body of experimental work (reviewed by Su¨dhof, 1995; Valtorta andBenfenati, 1995; Benfenati  et al. , 1999; Valtorta  et al. , 2001).Although a large number of proteins involved in exocytosishave been identified and many interactions among themhave been characterized in vitro, the precise physiologicalrole(s) of most of them have not yet been clearly demon-strated.A typical example is synaptophysin I (SypI), one of thefirst SV proteins to be identified ( Jahn  et al. , 1985; Wieden-mann and Franke, 1985). SypI is an abundant SV proteincharacterized by four membrane-spanning domains (Buck-ley  et al. , 1987; Leube  et al. , 1987; Su¨dhof   et al. , 1987). In vitro,SypI has been shown to form homo-oligomers composed of a variable number of subunits ( Jahn  et al. , 1985; Rehm  et al. ,1986) that, when incorporated into lipid bilayers, form volt-age-dependent channels with a conductance similar to thatof gap junctions (Thomas  et al. , 1988). Although SypI and thegap junction protein connexin share little sequence homol-ogy, the two proteins have similar membrane topologies andamino acid compositions of the third transmembrane do-main, which, in connexin, lines the gap junction pore (Leube,1995).Apparently contradictory data have been reported con-cerning the role of SypI in neurotransmitter release. Anti-sense oligonucleotides or antibodies directed against SypIdrastically reduced evoked release reconstituted in  Xenopus oocytes (Alder  et al. , 1992a; Shibaguchi  et al. , 2000). Consis-tently, antibodies to SypI reduced, and SypI overexpressionenhanced, acetylcholine release from  Xenopus  motor spinalneurons (Alder  et al. , 1992b, 1995). In contrast, SypI overex-pression decreased the secretion of growth hormone trans-fected in PC12 cells (Sugita  et al. , 1999). SypI knockout miceexhibited an apparently normal phenotype (Eshkind and Article published online ahead of print. Mol. Biol. Cell 10.1091/mbc.E02–01–0036. Article and publication date are at www.molbi-olcell.org/cgi/doi/10.1091/mbc.E02–01–0036. ‡ Corresponding author. E-mail address: valtorta.fl[email protected] used: Cyt, cytoplasmic; DIV, days in vitro; ECFP,enhanced cyano fluorescent protein; EYFP, enhanced yellowfluorescent protein; FRET, fluorescence resonance energy trans-fer; KRH, Krebs-Ringer-HEPES;  -Ltx,  -latrotoxin; SNARE, sol-uble  N  -ethylmaleimide-sensitive factor attachment protein re-ceptor; SV, synaptic vesicle; SV2, synaptic vesicle protein 2;SypI, synaptophysin I; SytI, synaptotagmin I; VAMP2, vesicle-associated membrane protein 2/synaptobrevin II.2706 © 2002 by The American Society for Cell Biology  Leube, 1995; McMahon  et al. , 1996), raising the possibilitythat other isoforms of the synaptophysin family (Knaus  etal. , 1990; Leube, 1994), or related proteins, such as the syn-aptogyrins (Baumert  et al. , 1990; Janz and Su¨dhof, 1998), cancompensate for the lack of SypI. Indeed, SypI/synaptogyrinI double knockout mice showed defects in both short- andlong-term potentiation ( Janz  et al. , 1999).SypI interacts in vitro with several SV proteins, includingthe v-soluble  N  -ethylmaleimide-sensitive factor attachmentprotein receptor (SNARE) vesicle-associated membrane pro-tein 2 (VAMP2)/synaptobrevin II (Calakos and Scheller,1994; Edelmann  et al. , 1995; Washbourne  et al. , 1995) as wellas with lipids, such as cholesterol (Thiele  et al. , 2000).VAMP2 is an integral SV protein (Baumert  et al. , 1989; Elf-erink  et al. , 1989), which interacts with the plasma mem- brane proteins syntaxin 1A and soluble  N  -ethylmaleimide-sensitive factor attachment protein-25 to form a complex (theSNARE complex) that drives fusion (So¨llner  et al. , 1993;reviewed by Pelham, 2001). Because the binding of VAMP2to SypI seems to be mutually exclusive with VAMP2 engage-ment in the SNARE complex (Edelmann  et al. , 1995), it ispossible that SypI, by sequestering VAMP2, impairs theassembly of SNARE complexes.The ability of SypI to interact with several SV constituentssuggests that it might be involved in multiple functionsduring the SV cycle. At all these sites, SypI does not seem toact alone but rather to cooperate with other proteins. Thiscould explain why SypI does not seem to be essential fortransmitter release but rather to participate in its regulation,playing either a positive (Alder  et al. , 1992a,b, 1995) or anegative (Sugita  et al. , 1999) role, depending on the systemand the experimental conditions investigated.In the present study, to overcome the limitations associ-ated with studying protein–protein interactions in vitro, wehave used, for the first time, video-enhanced microscopy of living neurons to detect fluorescence resonance energytransfer (FRET) between fluorescent SypI and VAMP2. Withthis technique, we have investigated the in vivo dynamics of SypI and VAMP2 homo-oligomerization and of SypI-VAMP2 interaction under resting conditions and duringexocytosis. MATERIALS AND METHODS Generation of Chimeric Fluorescent Proteins Rat SypI full-length cDNA (921 base pairs) cloned into the pBlue-Script vector (Stratagene, La Jolla, CA) was provided by Dr. R.Leube (University of Mainz, Mainz, Germany). SypI cDNA wasamplified by polymerase chain reaction (PCR) with the followingoligonucleotides: forward, 5  -GGGGGAAGCTTCAGCAGCAATG-GACGTG-3  ; and reverse, 5  -GGGGGGATCCGCTGCTGTAGTAG-CAGTAGGTCTTGGGCTCCACGCCCTTCATCTGATTGGAGAA -GGAGGTGG-3  .  Hin dIII and  Bam HI restriction sites, introducedwith the forward and reverse primers, respectively, are underlined.The reverse primer was designed to remove the stop codon and, inaddition, to introduce a linker of 13 amino acids (KGVEPKTY-CYYSS) (Nakata  et al. , 1998) at the COOH-terminal end of SypIcDNA. The resultant  Hin dIII/ Bam HI PCR fragment was insertedinto the corresponding sites of pECFP-N3 and pEYFP-N3 vectors(CLONTECH, Palo Alto, CA).The COOH-terminal deletion mutant of SypI (702 base pairs),lacking the last 73 amino acids of the protein and fused to theenhanced green fluorescent protein (EGFP) in the pEGFP-N3 vector(CLONTECH), was also provided by Dr. R. Leube. The mutatedSypI cDNA was digested with  Bam HI and BsrGI to remove EGFPand replace it with  Bam HI/BsrGI ECFP and EYFP fragments of pECFP-N3 and pEYFP-N3.VAMP2 full-length cDNA (351 base pairs) cloned into pBlueScriptwas from Drs. C. Montecucco and O. Rossetto (University of Padua,Italy). VAMP2 cDNA was amplified by PCR with the followingoligonucleotides: forward, 5  -GGGGTGTACAAGATGTCGGC-TACCGCTGCCAC-3  ; and reverse, 5  -GGGGGCGGCCGCTTA-AGTGCTGAAGTAAAC-3  . BsrGI and  Not I restriction sites, intro-duced with the forward and reverse primers, respectively, areunderlined. The resultant BsrGI/ Not I PCR fragment was insertedinto the corresponding sites of pECFP-N3 and pEYFP-N3.Synaptotagmin I (SytI) full-length cDNA (1265 base pairs) wassupplied by Dr. G. Schiavo (Imperial Cancer Research Fund, Lon-don, United Kingdom). After removing the stop codon by PCR, thecDNA was fused to the NH 2 -terminal end of EYFP in pEYFP-N3,generating the pSytI-EYFP vector. Cell Cultures and Transfections Transfection of Cos-7 cells was performed using a standard Ca 2  -phosphate precipitation protocol (Kingston, 1997). Cells were used72 h after transfection.Low-density, primary cultures of hippocampal neurons were pre-pared from Sprague-Dawley E18 rat embryos (Charles River Italica,Calco, Italy) as described previously (Banker and Cowan, 1977). Neurons were transfected at 3 d in vitro (DIV) by using 25-kDapolyethylenimine (PEI 25) (Sigma-Aldrich, Steinheim, Germany).Fresh medium was applied to cell cultures 1 h before starting theprocedure. Then, PEI 25 (28 nmol/dish) and plasmid DNA (2.5  g/dish) were diluted in 50   l of 150 mM NaCl in separate tubes.The solution containing PEI 25 was added to that containing theDNA, and the mixture was vortexed four times within 12 min before addition to the cells. Coverslips were placed in a clean 35-mmPetri dish and cells were rinsed with minimal essential mediumsupplemented with 10% horse serum, 2 mM glutamine, and 3.3 mMglucose. The medium was removed and cells were incubated for 2 hat 37°C in a 5% CO 2 , humidified atmosphere with 1 ml of the samemedium containing the 100   l of PEI 25/DNA solution. Coverslipswere then repositioned above astrocyte monolayers in the srcinaldishes and kept in culture for 15–18 d. Transfection efficiency variedfrom 0.1 to 1%.  Immunoblot Analysis Gel electrophoresis and immunoblotting of cell lysates were carriedout as described previously (Menegon  et al. , 2000) with either mono-clonal (R. Jahn, Max Planck Institute of Biophysical Chemistry, Go¨ttin-gen, Germany) or polyclonal (Valtorta  et al. , 1988) anti-SypI antibodies(1:5000 and 1:3000, respectively), polyclonal anti-VAMP2 antibody (1:500) (C. Montecucco), or monoclonal anti-GFP antibody (Roche Mo-lecular Biochemicals, Indianapolis, IN).  Immunofluorescence Analysis Immunofluorescence was performed as described previously (Me-negon  et al. , 2000), using the following primary antibodies: mono-clonal anti-synaptic vesicle protein 2 (SV2) (1:50) (K. Buckley, Har-vard University, Boston, MA), anti-microtubule associated protein-2(MAP2) (1:1000) (Roche Molecular Biochemicals) and polyclonalanti-SypI or anti-synapsin I (1:100) (Valtorta  et al. , 1988). In someinstances, primary antibodies were applied to unfixed cells inKrebs-Ringer solution buffered with HEPES (150 mM NaCl, 5 mMKCl, 1.2 mM MgSO 4 , 1.2 mM KH 2 PO 4 , 2 mM CaCl 2 , 10 mM glucose,and 10 mM HEPES/Na, pH 7.4) supplemented with 2 mM EGTA(KRH/EGTA). The incubation was carried out for 1 h at 37°C in 5%CO 2 . After two washes with KRH/EGTA, samples were fixed andprocessed for indirect immunofluorescence. Images were recordedwith a C4742-98 ORCA II cooled charge-coupled device cameraAnalysis of Synaptophysin I InteractionsVol. 13, August 2002 2707  (Hamamatsu Photonics, Hamamatsu City, Japan) and processedusing the computer program ImagePro Plus 4.0 (Media Cybernetics,Silver Spring, MD). Spectrofluorometric Analysis Cos-7 cells were transiently transfected with an expression vectorencoding one of the chimeric, fluorescent proteins of interest. Sev-enty-two hours after transfection, the cells were washed twice withphosphate-buffered saline and collected by scraping. The cells werethen pelleted by centrifugation, resuspended in 700  l of phosphate- buffered saline, and analyzed in a spectrofluorometer (LS50B;PerkinElmer, Shelton, CT).  FM1-43 Assay FM1-43 (8   M) (Molecular Probes, Eugene, OR) was loaded intorecycling SVs of 16 DIV hippocampal neurons by using a depolar-izing solution containing KRH supplemented with 45 mM KCl and10   M 6-cyano-2,3-dihydroxy-7-nitroquinoxaline. The incubationwas carried out for 90 s at room temperature and was followed byrinsing for 15 min with a 2-ml/min flow of KRH containing6-cyano-2,3-dihydroxy-7-nitroquinoxaline. After the washing proto-col, images were recorded using fluorescein excitation and rhoda-mine emission filters, and a 40   oil immersion objective. Averageintensities for each bouton (I KRH ) were measured. Cells were thenrapidly rinsed with KRH/EGTA and incubated for 40 min at roomtemperature in the same solution in the absence or presence of 0.1nM   -latrotoxin (  -Ltx) (A. Petrenko, New York University MedicalCenter, New York, NY). After 15 s of continuous illumination forfocusing on the specimen, a series of 20 images at 6-s intervals wererecorded for each of the fields previously acquired. FM1-43 releasewas calculated by comparing the intensity of fluorescence in eachsynaptic bouton before and after the incubation. To correct for thereduction in fluorescence intensity due to photobleaching that oc-curred during the 15-s exposure used for focusing, an exponentiallydecaying curve of the form y(t)  Ae  t/    C was fit to the averageintensity vs. time data for each single synaptic bouton in the se-quence of images acquired. This expression was used to calculate acorrected postincubation fluorescence intensity (I KRH/EGTA ) for each bouton. FM1-43 release was then calculated as (1    I KRH/EGTA /I KRH ).  -Latrotoxin Binding Assay Anti   -Ltx antibody was purchased from Alomone Laboratories(Jerusalem, Israel), conjugated to Cy3 (Amersham Biosciences, Pis-cataway, NJ), and purified according to the manufacturer’s instruc-tions. Hippocampal neurons of 16 DIV were washed once withKRH/EGTA and incubated for 30 min at 37°C in 5% CO 2  in thesame solution supplemented with Cy3-conjugated, anti   -Ltx anti- body (50   g/ml) in the absence or presence of 0.1 nM   -Ltx. Thecells were washed twice with KRH/EGTA, and Cy3 images wereacquired with a standard Texas Red filter set.  Fluorescence Resonance Energy Transfer (FRET) Analysis Expression vectors encoding fluorescent proteins were cotrans-fected at a ratio of 1:2 or 1:4 (donor/acceptor). Cells (15–18 DIV)were washed once with KRH/EGTA and incubated in the samesolution either in the presence or absence of 0.1 nM   -Ltx for 30 minat 37°C in 5% CO 2 ; the cells were then washed twice with KRH/EGTA. Images were acquired within 30–45 min after treatment of the cells. The specimen was irradiated at the wavelength of 436  10nm, and a time-lapse series of images of the donor fluorescencewere recorded at the wavelength of 480  30 nm during continuousillumination. From the first image of the series, a binary mask wasprepared, in which each spot corresponded to a synaptic bouton.Fluorescent spots that moved quickly along the axon (and thatpresumably represented traveling packets) were excluded from theanalysis. The time series data for each pixel position within a boutonwere fit to an exponential decay function to determine decay con-stants of photobleaching (Figure 1).When FRET occurs between donor and acceptor fluorophores, thetime constant for donor photobleaching increases (Jovin and Arndt- Jovin, 1989). Thus, the efficiency (E) of FRET was calculated as thepercentage of change in the average time constant of donor photo- bleaching measured in specimens transfected with the SV-locatedacceptor fluorescent proteins (   sv*sv ), with respect to that measuredin specimens transfected with cytosolic EYFP acceptor (   sv*cyt ) viathe following equation: E  1  (   sv*cyt /   sv*sv ).One of the advantages of this method for measuring FRET is thatthe measurements do not depend on absolute values of fluores-cence. Indeed, we found no significant correlation between initialintensities of fluorescence and photobleaching rates (R    0.4). Thephotobleaching time constants were found to have skewed distri- butions, which became normal after logarithmic transformation.Therefore, data were analyzed using the natural logarithms of thephotobleaching time constants, and efficiencies and statistics werederived by retransformation of the pertinent values. Where indi-cated, one-tailed  t  tests were performed to estimate the significanceof differences between mean FRET efficiencies. To estimate theprobability that a given mean FRET efficiency was statistically dif-ferent from zero, the mean value normalized by the SD of the meanwas compared with a one-tailed Z distribution. RESULTS Generation and Characterization of Chimeric Fluorescent Proteins SypI and VAMP2 To apply the FRET technique to the study of the molecularinteractions occurring during exocytosis, we fused ECFP orEYFP to the SV proteins SypI and VAMP2. The fluorescentproteins were fused to the cytosolic, COOH-terminal tail of SypI, to obtain SypI-ECFP and SypI-EYFP, or to the cytoso- Figure 1.  Time course of the decay of donor fluorescence duringcontinuous illumination. The figure shows an example of the fluo-rescence intensity of SypI-ECFP transfected in hippocampal neu-rons measured in a series of time-lapse images by using a 12-bitmonochrome video camera. Measurements were carried out sepa-rately for individual pixels in one field of view (total number of pixels, 634). The intensity values at each time point were averagedand plotted.M. Pennuto  et al .Molecular Biology of the Cell2708  lic, NH 2 -terminal end of VAMP2 to generate ECFP-VAMP2and EYFP-VAMP2. Chimeras of a SypI deletion mutantlacking the cytosolic, COOH-terminal tail of the protein(SypI  C-ECFP and SypI  C-EYFP) were also prepared. Inaddition, EYFP was fused to the cytosolic, COOH terminusof SytI, to generate SytI-EYFP.The expression of the full-length fusion proteins was ver-ified in non-neuronal Cos-7 cells transfected with the appro-priate vectors (Figure 2A; our unpublished data). In addi-tion, the fusion proteins were shown to exhibit spectralproperties similar to those of the soluble fluorophores(Tsien, 1998; Figure 2B; our unpublished data).Hippocampal neurons were transfected at 3 DIV and keptin culture until 15–18 DIV, which corresponds to full matu-ration and the establishment of a synaptic network withsurrounding cells (Valtorta and Leoni, 1999). We verified theexpression and proper targeting of the chimeras, as well asthe absence of toxicity related to the sustained, high level of expression. Immunolabeling of neurons cotransfected withthe expression vectors encoding ECFP-VAMP2 and SypI-EYFP confirmed that both fusion proteins colocalized withthe endogenous SV protein SV2 (Bajjalieh  et al. , 1994; Figure2C; our unpublished data). Indeed, the colocalization coef-ficients of SV2 with ECFP-VAMP2, SypI-ECFP, or SytI-ECFPwere 0.88, 0.80, and 0.99, respectively. Furthermore, theexogenous proteins were delivered to axons and did notcolocalize with MAP2, which in mature neurons is presentexclusively in the somatodendritic compartment (Kosik andFinch, 1987).No apparent developmental changes due to overexpres-sion of the transfected proteins could be detected. In partic-ular, there were no effects on the density of synapses (4.6  2.4 and 3.8    0.8 synapses/10-  m neurite length in theuntransfected and transfected neurons, respectively) nor onthe number of synaptic vesicles per terminal (our unpub-lished data). Effect of    -Ltx on Synaptic Boutons To trigger exocytosis, hippocampal neurons at 15–18 DIVwere treated with 0.1 nM purified   -Ltx for 30 min in Ca 2  -free medium (KRH/EGTA), a condition known to causemassive exocytosis of SVs in the absence of endocytosis(Ceccarelli and Hurlbut, 1980; Valtorta  et al. , 1988). Videoanalysis showed that, after a 10-min delay, the morphologyof the axons changed progressively, and at the end of thetreatment the axons assumed a characteristic bead-shaped Figure 2.  Expression and targeting of the SypI and VAMP2 fluo-rescent fusion proteins. (A) Fusion proteins are properly translatedin the cells. Cos-7 cells were transiently transfected with the expres-sion vectors encoding either SypI-ECFP (lanes 1 and 3), SypI-EYFP(lanes 2 and 4), ECFP-VAMP2 (lanes 5 and 7), or EYFP-VAMP2(lanes 6 and 8). Seventy-two hours after transfection, cells wereanalyzed by immunoblotting with anti-green fluorescent protein(lanes 1, 2, 5, and 6) and either anti-SypI (lanes 3 and 4) or anti-VAMP2 (lanes 7 and 8) antibodies. (B) Fusion of the fluorescentproteins to the SV proteins does not alter the spectral properties of the fluorophores. The excitation (solid) and emission (dashed) spec-tra of the chimeras were measured in suspensions of transientlytransfected Cos-7 cells. (C) Exogenous SV fusion proteins are tar-geted to synaptic boutons in transfected neurons. Hippocampalneurons were cotransfected with the expression vectors encodingSypI-EYFP and ECFP-VAMP2 and processed for immunofluores-cence with either anti-SV2 or anti-MAP2 antibodies. (a–d) Colocal-ization of both ECFP-VAMP2 (a) and SypI-EYFP (b) with SV2 (c),and overlay of a, b, and c (d). (e–h) Lack of colocalization of ECFP-VAMP2 (e) and SypI-EYFP (f) with MAP2 (g), and overlay of e, f, and g (h). Bar, 10   m.Analysis of Synaptophysin I InteractionsVol. 13, August 2002 2709
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