Synaptotagmin: a Calcium Sensor on the Synaptic Vesicle Surface Author(S): Nils Brose, Alexander G. Petrenko, Thomas C. Südhof

Home , Synaptic vesicle, Synaptotagmin

Synaptotagmin: A Calcium Sensor on the Synaptic Vesicle Surface Author(s): Nils Brose, Alexander G. Petrenko, Thomas C. Südhof and Reinhard Jahn Source: Science, New Series, Vol. 256, No. 5059 (May 15, 1992), pp. 1021-1025 Published by: American Association for the Advancement of Science Stable URL: http://www.jstor.org/stable/2877129 Accessed: 01-10-2015 19:18 UTC

Your use of the JSTOR archive indicates your acceptance of the Terms & Conditions of Use, available at http://www.jstor.org/page/ info/about/policies/terms.jsp

JSTOR is a not-for-profit service that helps scholars, researchers, and students discover, use, and build upon a wide range of content in a trusted digital archive. We use information technology and tools to increase productivity and facilitate new forms of scholarship. For more information about JSTOR, please contact [email protected].

American Association for the Advancement of Science is collaborating with JSTOR to digitize, preserve and extend access to Science. http://www.jstor.org

This content downloaded from 132.239.70.252 on Thu, 01 Oct 2015 19:18:21 UTC All use subject to JSTOR Terms and Conditions . REPORTS

that does not require calculation of derivatives is placed by two dots (situated at the endpoints). Weinshall, Biol. Cybern. 64, 209 (1991). to look for random changes (controlled in appro- The network learned this task, as it did previously 19. S. Edelman, D. Reisfeld, Y. Yeshurun, CS-TR priate ways) in the parameter values that reduce in the line vernier and the bisection cases. The 91-20 (Department of Applied Mathematics and the error. In the simulations described in this better performance of the HyperBF module in the Computer Science, Weizmann Institute, Rehovot, report the model was endowed with a dual, incre- dot vernier task for small offsets parallels a recent Israel, 1991); R. Brunelli and T. Poggio, in Pro- mental learning mechanism. First, when the mod- surprising finding with human subjects (M. Fahle, ceedings of the 12th International Joint Confer- el's performance on a new input was markedly unpublished observations). ence on Artificial Intelligence, International Joint inadequate (in comparison with recent history), 13. In a recent study, R. Bennett and G. Westheimer Conference on ArtificialIntelligence, Inc., Sydney, that input was adjoined to the model as an addi- [Percept. Psychophys. 49, 541 (1991)] found sur- Australia, 24 to 30 August 1991 (Kaufmann, tional center (prototype). This happened mainly in prisingly little learning of thresholds in three-dot Mountain View, CA, 1991); S. Edelman and T. the initial trials, with the number of centers even- alignment and grating discrimination. Their exper- Poggio, A.I. Memo No. 1181 (ArtificialIntelligence tually reaching an asymptote that depended on iments used transfer of training across the stimu- Laboratory, Massachusetts Institute of Technolo- the nature of the task and on the parameters that lus range to probe for learning, hiding possible gy, Cambridge, MA, 1990); R. Brunelli and T. affected the decision to add new centers. The effects of fast learning that may have happened in Poggio, I.R.S. T. TechReport 9110-04 (Instituto per performance of the model during these first trials the baseline session (p. 544). Interestingly, the la Ricerca Scientifica e Tecnologica, Trento, Italy, improved quickly, then stabilized as the number lack of transfer across the stimulus range in these 1991). of centers approached the asymptote. Second, experiments is consistent with our notion of expe- 20. We are grateful to H. Buelthoff, F. Crick, F. Girosi, further gradual improvement in the performance rience-based learning. R. Held, A. Hurlbert,Y. Weiss, and G. Westheimer was obtained by letting the model carry out a local 14. A. Fiorentini and N. Berardi, Nature 287, 43 for useful discussions and suggestions. Support- random search in the space of existing HyperBF (1980). ed by a grant from the Office of Naval Research, center coordinates. This search was guided by 15. A. Karniand D. Sagi, Proc. Natl. Acad. Sci. U.S.A. Cognitive and Neural Sciences Division, by the feedback given to the model (that is, by indicating 88, 4966 (1991). Artificial Intelligence Center of Hughes Aircraft whether the response at each trial was correct). 16. K. Ball and R. Sekuler, Science 218, 697 (1982); Corporation, and by the Deutsche Forschungsge- Details of the learning algorithms, including an S. P. McKee and G. Westheimer, Percept. Psy- meinschaft (Heisenberg-Programme). Support for extension of the incremental learning algorithm to chophys. 24, 258 (1978); V. S. Ramachandran the Artificial Intelligence Laboratory's artificial in- a situation in which no explicit feedback is avail- and 0. Braddick, Perception 2, 371 (1973). telligence research is provided by the Advanced able, can be found in (6, 7). 17. Y. Fr6gnac, D. Shulz, S. Thorpe, E. Bienenstock, Research Projects Agency of the Department of 5. A description of multilayer perceptrons and the Nature 333, 367 (1988). Defense. T.P. is supported by the Uncas and back-propagation technique used for learning is 18. T. Poggio and S. Edelman, ibid. 343, 263 (1990); Helen Whitaker chair. in D. E. Rumelhart, G. E. Hinton, R. J. Williams, S. Edelman and T. Poggio, Int. J. Pattern Recog- Nature 323, 533 (1986). An overview of some of nit. Artif. Intell., in press; S. Edelman and D. 18 December 1991; accepted 24 March 1991 the classical techniques can be found in S. Omo- hundro [Complex Syst. 1, 273 (1987)] and in R. 0. Duda and P. E. Hart [Pattern Classification and Scene Analysis (Wiley, New York, 1973)]. Rela- tions between multilayer perceptrons and Hy- Synaptotagmin: A Calcium Sensor on the perBF networks are mentioned in (4) and studied in M. Maruyama, F. Girosi, T. Poggio, A.l. Memo Synaptic Vesicle Surface No. 1291 (Artificial Intelligence Laboratory, Mas- sachusetts Institute of Technology, Cambridge, MA, 1992). Nils Brose,* Alexander G. Petrenko, Thomas C. Sudhof, 6. T. Poggio, M. Fahle, S. Edelman, A.l. Memo No. ReinhardJahntt 1271 (Artificial Intelligence Laboratory, Massa- chusetts Institute of Technology, Cambridge, MA, Neurons release neurotransmitters by calcium-dependent exocytosis of synaptic 1991); S. Edelman, T. Poggio, M. Fahle, Comput. vesicles. Vision Graph. Image Process. B, in press. The However, the molecular steps transducing the calcium signal into membrane fusion are still simulation results were robust with respect to all an enigma. It is reported here that synaptotagmin, a highly conserved synaptic vesicle parameters, including the number of inputs. protein, binds calcium at physiological concentrations in a complex with negatively charged 7. Y. Weiss, S. Edelman, M. Fahle, T. Poggio, CS-TR 91-21 (Department of Applied Mathematics and phospholipids. This binding is specific for calcium and involves the cytoplasmic domain of Computer Science, Weizmann Institute, Rehovot, synaptotagmin. Calcium binding is dependent on the intact oligomeric structure of syn- Israel, 1991). aptotagmin (it is abolished by proteclytic cleavage at a single site). These results suggest 8. We have also experimented with a different ver- that synaptotagmin sion of the HyperBF model, in which orientation- acts as a cooperative calcium receptor in exocytosis. selective receptive fields similar to those of simple cells in Vi played the role of the basis functions. See (7). This version of the model replicated the absolute values and the time course of the improvement of the thresholds found in human psy- Calcium-dependentexocytosis of synaptic entryand the releaseof transmitteris in the chophysical data, in addition to replicating the vesicles is the central step in the sequence range of 200 ,us. This implies that a com- data concerning the percentage of correct re- of events from the arrival of an action plex betweensynaptic vesicles and the plas- sponses. 9. Hyperacuity-level performance was independent potential to the release of neurotransmit- ma membranemust exist in the restingstate of the precise location of the receptors. At the ters. It is generally accepted that Ca2+ because the time after Ca2+ entry is too same time, different quasi-random receptor mo- enters the nerve terminalvia voltage-gated short to allow for vesicle docking before saics yielded different thresholds, sometimes by Ca2+ as much as a factor of 2. A similar range of channels in the presynapticplasma fusion. Furthermore,the dependence of hyperacuity thresholds is observed in human sub- membrane.Intracellular recordings in mod- transmitterrelease on the intraterminal jects, even at full acuity and with perfectly normal el synapsessuch as the squidgiant synapse Ca2+ concentrationis nonlinearand highly eyes. have shown that the latencybetween Ca2+ cooperative(1). 10. The model also exhibited learning on a longer time scale (4, 7), similar to the slow long-term The Ca2+ receptorprotein for exocytosis learning component found in human subjects (M. N. Brose and R. Jahn, Department of Neurochemistry, has not been identified. However, certain Max-Planck-Institute for Fahle and S. Edelman, in preparation). Psychiatry, D-8033 Martins- predictions about its properties can be 11. R. Watt and F. W. Campbell, Spat. Vision 1, 31 ried, Germany. A. G. Petrenko and T. C. SOdhof, Howard Hughes (1985). made. Becauseof the shortlatency between Medical Institute and Department of Molecular Genet- 12. The stimulus in the bisection task consists of three Ca2+ influx and exocytosis,it is likely that ics, University of Texas Southwestern Medical Center, dots, arranged in a vertical line, at an approxi- Dallas, TX 75235. the Ca2+ receptoris part of the complex mately even spacing. The subject has to deter- formed mine whether the middle dot is above or below the *Present address: Salk Institute, Molecular Neurobiol- between the plasmamembrane and midpoint of the segment formed by the other two ogy Laboratory, 10010 North Torrey Pines Road, La the synapticvesicle and is probablylocated dots. The HyperBF module learned this hypera- Jolla, CA 92037. on one of these membranecompartments. cuitytask just as easily as it did in the linevernier tPresent address: Howard Hughes Medical Institute In addition,Ca2+ must induce a change in case (6). Anothersimulation made a comparison and Department of Pharmacology, Yale University between the line verniertask and a similarone in School of Medicine, New Haven, CT 06536. the properties of the receptor protein, which each of the line segments has been re- fTo whom correspondence should be addressed. which ultimatelycauses a rearrangementof

SCIENCE * VOL. 256 * 15 MAY 1992 1021

This content downloaded from 132.239.70.252 on Thu, 01 Oct 2015 19:18:21 UTC All use subject to JSTOR Terms and Conditions phospholipidmicrodomains at the contact tence of two copies of an internal repeat rected against synaptotagminI as affinity site betweenthe two membranes,leading to that are homologous to corresponding do- ligand (6). SynaptotagminI was purifiedin membranefusion. It is unclearhow many mains in protein kinase C (C2 domain) (3, a single step, resultingin a proteinof more proteinsparticipate in this process.Howev- 4), in a cytosolic form of phospholipase than 90% purity. er, the speedof the event makesit unlikely A2, and, although with less identity, in We studiedthe Ca2+ bindingto purified that majorprotein rearrangements, such as guanosine triphosphatase-activating pro- synaptotagminby equilibriumdialysis ei- Ca2+-induced docking of cytosolic protein and phospholipase C (5). In protein ther in the presence or in the absence of teins, are involved (1). kinase C and phospholipase A2, this do- phospholipids(7). As controls,Ca2+ bind- In this report,we presentevidence that main is thought to be responsible for the ing to phospholipidsalone or to purified the synapticvesicle proteinsynaptotagmin Ca2+-dependent binding of these proteins synaptophysin,an integralmembrane pro- (also referredto as p65) exhibits properties to phospholipid membranes (5). This tein of synapticvesicles (8), was monitored expected of the exocytotic Ca2+ receptor. prompted us to investigate whether native under identical experimental conditions. Synaptotagminis representedby a family synaptotagmin functions as a Ca2+ bind- No Ca2+ binding to synaptotagminwas of relatedintegral membrane proteins with ing protein and whether Ca2+ binding by observedin the absence of phospholipids widespreaddistribution (2-4). It is specif- synaptotagmin involves interactions with (Fig. 1). However, when liposomes con- ically localizedin the membraneof synap- membrane phospholipids. taining 25% phosphatidylserineand 75% tic vesicles and that of secretorygranules To study the properties of synaptotag- phosphatidylcholinewere added,a dramat- in endocrine cells (2). Structuralanalysis min, we isolated the protein from Triton ic increasein Ca2+ bindingover that of the of this protein family revealed the exis- X-100 extracts of rat brain by affinity chro- phospholipidcontrol was observed(Fig. 1). matography, using a newly generated The Ca2+ bindingreached 4 mol per mole monoclonal antibody (clone Cl 41.1) di- of synaptotagminsubunit at a free Ca2+ 10- A 9- Ca2fe &tM) B A w 15

0 7C- C-O E 47 3. I? I 10

w i c 2- Z ~I/ 40- 5-

o:= 0 ,0 L >l O , _iv- 0 -8 -7 -6 -5 -4 5- B L s @ ~~~~~~~~~~~~-9-8 -7 -6 -5 -4 -3 4- _~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~I I a. 5 0~~~~5 Time (min) C ,E 3 - Fig. 2. Ca2+-dependentinteraction of purified o synaptotagmin with phospholipid vesicles, r. 0 2 measured by fluorescence resonance energy + transferbetween tryptophanresidues of phos- 3 - pholipids(excitation at 284 nm)and dansylated Cl~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~phospholipid head groups (emission at 520 0~~~~~~ nm). Synaptotagminwas added eitherin micel- 2 - lar form (A and B) or incorporatedinto liposomes (C). (A) Ca2+ causes an increase of fluorescence resonance energy transfer,which 1 - -8 -7 -5 -6 -4 is reversible upon EGTAaddition. The phos- log Ca2+fme pholipidcomposition of the liposomes was 50% 0 Fig. 1. Binding of Ca2+ to synaptotagmin in the phosphatidylserine, 40% phosphatidylethanol- presence or absence of phospholipid vesicles, amine, and 10% dansyl-phosphatidylethanol- I 8 I - i determined by equilibrium dialysis (7). As con- amine (13). (B) Ca2+-dependentfluorescence -9 -8 -7 -5 -4 -3 trol, Ca2+ binding to protein-free liposomes is resonance energy transfer is saturable and log Ca2fr, shown. (A) Total Ca2+ binding: (@) synaptotag- depends on the phospholipidcomposition. Values were expressed as a percentage change in min plus phospholipid vesicles; (0) phospho- emission intensity,/net = (/ - /0) x 100/10,where / is the recorded fluorescence intensityin the lipid vesicles alone; (V) synaptotagmin alone. experimentand /1 is the reference fluorescence intensityin the absence of Ca2+. (-) Liposomes Note that phospholipid vesicles bind Ca2+ in a containing 50% phosphatidylserine,40% phosphatidylethanolamine,and 10% dansyl-phosphati- linear dependence on the Ca2+ concentration. dylethanolamine(mean values of three independent experiments);(-) liposomes containing25% (B) Net Ca2+ binding to synaptotagmin (-) phosphatidylserine,65% phosphatidylethanolamine,and 10% dansyl-phosphatidylethanolamine; and, as control, to synaptophysin (V), in the (V) control,using an assay mixtureas in (-) that was digested for 1 hour at 37?Cwith trypsin(1 presence of phospholipids. We obtained the iLg/ml)before the experiment (14) (data shown are from a representative experiment). (C) values by subtractingbinding to phospholipids Ca2+-dependent fluorescence resonance energy transferbetween synaptotagmin-containingdo- alone from total binding. Numbers of moles nor liposomes and dansylated acceptor liposomes. The donor liposomes were composed of 45% were calculated on the basis of the molecular phosphatidylcholine,45% phosphatidylethanolamine,and 10% phosphatidylserine;the acceptor weight of the monomers,respectively. liposorneswere the same as in (A).

1022 SCIENCE * VOL. 256 * 15 MAY 1992

This content downloaded from 132.239.70.252 on Thu, 01 Oct 2015 19:18:21 UTC All use subject to JSTOR Terms and Conditions .... --11111.1-E-PORT-S concentrationof 10-4 M (Fig. 1). In con- Flg. 3. Radiolabeled liposomes bind reversibly trast, no significantCa2+ binding was ob- to synaptotagmin immobilized to protein G- served up to a concentration of 1o-4 M Sepharose. Purified synaptotagmin (10 Lg), when purifiedsynaptophysin instead of syn- suspended in 50 ILI of 20 mM tris-CI (pH 7.2), , 10- E aptotagminwas used, regardlessof whether 100 mM NaCI was incubated with 20 9g of Ca2+ phospholipidswere 1). purified monoclonal antibody to synaptotagmin 2 present (Fig. This for 1 hour at 4?C and bound to protein G- 8- indicates that, contraryto an earlierreport Sepharose by further incubation for 1 hour (8-p.l 0 (8), synaptophysindoes not bind Ca . bed volume). Additions of Ca2+ (left pair of 0 Ca 2+ Thus, the abilityto bind Ca2+ is a specific, bars) or EGTA (middle pair of bars) were made 6 intrinsicproperty of synaptotagmin. to give final concentrations of 1 mM. Binding - To studythe interaction was started by the addition of 100 Il (500 9g) g EGTA EGTA betweensynap- 4 totagminand phospholipidsin moredetail, of 14C-labeled liposomes, prepared from 50% - we utilizeda fluorescenceresonance energy phosphatidylserine and 50% phosphatidyletha- transferassay. nolamine, and carried out for 10 min. To some z 2 Dansyl-phosphatidylethanol- co amine was incorporatedinto liposomes. Ca2+-containing samples, EGTAwas then added to give a final concentration of 1.5 mM and These liposomeswere mixed with purified X\ the incubation was continued for 5 min (right a 0 synaptotagmin.Fluorescence resonance en- pair of bars). The reaction was stopped by ergy transferbetween tryptophanresidues removal of the assay mixture from the beads by filtration.The beads were rapidly washed twice with in synaptotagminand the dansyl group in incubation buffer containing 1 mM Ca2+ (left pair of bars) or 1 mM EGTA (middle and right pairs of the liposomeswas measuredas a functionof bars) and analyzed by liquid scintillation counting. Hatched bars show background binding of Ca2 . Energytransfer is highly dependent liposomes to protein G-Sepharose containing monoclonal antibody but no synaptotagmin. on the distance between the two fluoro- phores and is only observedon close contact of the proteinand the lipid fluorescent concentration was remarkablysimilar to vesicles and the plasmamembrane. probe. We found that Ca2+ triggeredfluo- that of Ca2" bindingmeasured by equilib- To ensure that synaptotagmincan also rescence resonance energy transfer in a riumdialysis, with a half-maximalresponse interact with phospholipidvesicles when dose-dependentmanner (Fig. 2A), suggest- at lo-5 M Ca2" (compareFigs. lB and the proteinitself is incorporatedin a phos- ing that Ca2+ causesa close associationof 2B). Although the precise phospholipid pholipid vesicle, we purifiedthe protein synaptotagminwith the dansyl liposomes. composition of the presynaptic plasma from cholate extracts and reconstitutedit The signals were reversedby addition of membraneis not known, a proportionof into liposomes by a dialysis procedure. EGTA (Fig. 2A) and reestablishedby sub- 25%acidic phospholipidsis probablya low When acceptorliposomes containing dan- sequent addition of excess Ca2+ (not estimate. Because an increase in acidic sylated head groups were added to these shown), demonstratingthat the interaction phospholipidcontent drasticallyshifts the liposomes, Ca2l-dependent fluorescence is reversible.Liposomes alone showed no Cal2+ sensitivity to lower concentrations, resonance energy transfer was observed, change in fluorescence on addition of the response lies well within the Ca2+ which was similarto that observedwith the Ca2+. Furthermore,no energytransfer was concentrationrange expected upon excita- purifiedprotein alone (Fig. 2C). As an observedup to a Ca2+ concentrationof 1 tion at the contact site between synaptic independentconfirmation for Ca2+-depen- mM when synaptotagminwas subjectedto mild proteolysis with trypsin before the experiment (Fig. 2B) or when equal amountsof purifiedsynaptophysin or purified immunoglobulinG (IgG) were used instead of synaptotagmin (not shown). '4, CHAPS SDS These controlsdemonstrate that this assay 213 14 15161718 | 910111112|13 5I16171811314 | 911011112113 measuresthe specific Ca2+-dependent in- -80 teractionof synaptotagminwith liposomes. p65 ",, ., Energytransfer between synaptotagmin |no -50 and liposomeswas specificfor Ca2+ (Mg2+, trypsn Ba2+, or Sr2+ did not evoke a responseat -B concentrationsup to 1 mM; these metal esm- ~ ions were also unable to interferewith the Ca2+ signal). However,an increasein both the Ca2+ sensitivity and the intensity of the signal was observedwhen the propor- NH2 !ermlnus- -_ tion of phosphatidylserinein the acceptor 80 liposomeswas increasedto 50% (Fig. 2B). + trypsin -50 This indicates that the acidic head groups C2 domains-- --- ,, may participatedirectly in the formation of the synaptotagmin-Ca2+-phospholipid complex, which is in agreementwith the Fig. 4. Mild proteolytic cleavage of synaptotagmin results in monomeric fragments containing the C2 previouslydetermined phospholipid bind- domains and oligomeric NH2-terminal fragments. Intact synaptotagmin (p65) or synaptotagmin cleaved at a single site by low doses of trypsin was solubilized from synaptic vesicles, and ing specificityof recombinantsynaptotag- its apparent size (Mr) was analyzed by sucrose gradient centrifugation in the presence of CHAPS (left) min (3). or SDS (right). Fractions were immunoblotted after SDS-PAGE with an antibody either to the 02 For a given phospholipidcomposition domains or to the NH2-terminus of synaptotagmin, and immunoreactive bands were visualized with (25% acidic phospholipids), the depen- chemoluminescence. Analysis of fractions in cholate or octylglucoside as detergents gave resufts dence of phospholipidbinding on the Ca2+ similar to those in CHAPS (not shown) (15).

SCIENCE * VOL. 256 * 15 MAY 1992 1023 This content downloaded from 132.239.70.252 on Thu, 01 Oct 2015 19:18:21 UTC All use subject to JSTOR Terms and Conditions dent interaction of synaptotagminwith trophoresis(SDS-PAGE) and immunoblot- totagmin (3) used as antigen. Preparation of the phospholipid vesicles, we measured affinity matrix (1-ml final volume, containing -8 the ting (3). mg of purified IgG) and purification of synaptotag- bindingof radiolabeledliposomes to immo- BecauseCa2+-phospholipid binding was min (and synaptophysin) were performed essen- bilizedsynaptotagmin. Whereas in the pres- abolishedby proteolyticcleavage at a single tially as described earlier for synaptophysin [F. ence of EGTA a small but significant site, the responsibilityof the C2 domains Navone et al., J. Cell Biol. 103, 2511 (1986)], by using a Triton X-100 extract of rat brain mem- amount of phospholipidbinding was ob- could not be unambiguouslyestablished branes as starting material. Protein was eluted in served, Ca2" induced a four- to sixfold althoughit seems likely in analogyto pro- 0.05% Triton X-100 and dialyzed for 2 days increaseover the basalbinding, which was tein kinase C and phospholipaseA2. The against six buffer changes before the experiments. Yields were between 0.2 and 0.4 mg of abolished upon subsequent addition of observationof fluorescenceresonance ener- protein per preparation. EGTA (Fig. 3). gy transferbetween tryptophanand phos- 7. Ca2+ binding was measured with an equilibrium In synaptic vesicles, synaptotagminis pholipidsstrongly supports an involvement dialysis assay (10). Dialysis samples contained 0.2 to 0.6 mg of protein (synaptotagmin or synap- present as a homo-oligomericcomplex of of the C2 domainsbecause these domains tophysin) per milliliteror 1.7 mg of phospholipid probablyfour subunitscontaining eight C2 contain the only tryptophanresidues of the vesicles (75% phosphatidylcholine, 25% phos- domains (9). To evaluate whether Ca2+- entire structure,the single exception being phatidylserine) per milliliter,which were prepared phospholipidbinding by synaptotagminis a tryptophanresidue in as described in (13). As an internal standard to located the mem- follow sample dilution during dialysis, liposomes dependent on an intact oligomeric struc- brane-spanningdomain, which is unlikely containing 3H-labeled phosphatidylcholine (6 ture, we subjectedthe protein to limited to be included. However, the resultsindi- ,uCi/mg), which do not bind synaptotagmin or proteolysis.Under these conditions, cleav- cate that an intact tetramericstructure of synaptophysin, were added to a final concentration of 0.33 mg/ml. Concentrations of free Ca2+ age occurs at a single site adjacentto the the protein is requiredfor Ca2+ binding. were adjusted in an EGTA-Ca2+ buffer system. membrane spanning domain, creating a This requirementand the presenceof mul- We calculated the EGTA and Ca2+ concentra- large cytoplasmicfragment that contains tiple Ca2+-bindingsites may explain the tions needed to give the desired free Ca2+ concentrations using computer software developed both C2 domains (3). This cleavage abol- high cooperativityobserved for Ca2+ in by K. J. Fohr, W. Warhol, and M. Gratzl (Methods ished the ability of synaptotagminto bind triggeringtransmitter release. Enzymol., in press). Samples were dialyzed for 12 Ca2+ or phospholipidsin a Ca2+-depen- How does synaptotagminfunction in hours against large volumes of [45Ca]Ca2+ containing buffer and then assayed for 45Ca and 3H dent manner as measuredwith the assays exocytosis?Although we have demonstrat- by scintillation counting. describedabove (Fig. 2B). To analyzethe ed Ca2+-dependent binding of synaptotag- 8. H. Rehm, B. Wiedenmann, H. Betz, EMBO J. 5, structureof the proteolytic fragmentsin min-containingmembrane vesicles to ac- 535 (1986). For a review on synaptophysin and more detail, we determinedtheir size by ceptor liposomes, we do not believe that other synaptic vesicle proteins, see also T. C. Sudhof and R. Jahn, Neuron 6, 665 (1991). sucrose density gradient centrifugationin this propertyis solelyresponsible for vesicle 9. Evidence that synaptotagmin is a tetramer: Using CHAPS. For comparison,the migrationof docking to the presynapticplasma mem- a variety of detergents, we found that synaptotag- the fragments was monitored in SDS, brane. Instead,we assumethat in the rest- min migrates at a similar position on sucrose gradients. This position is different from that of which dissociatesall aggregatesinto mono- ing nerve terminalwhere the Ca2+ concen- synaptophysin, synaptobrevin, synapsin, synap- mers. The NH2-terminalfragment of syn- tration is low (1), synaptotagminforms a toporin, and the proton pump, suggesting that aptotagmincomigrates with uncleavedsyn- complexwith a specificacceptor protein in synaptotagmin is not part of an artifactual high molecular weight complex due to insufficient sol- aptotagmin in a high molecular weight the plasma membrane.We have recently ubilization (3). In the presence of Zwittergent complex (Fig. 4), clearlyseparated from the reportedthat synaptotagminbinds specifi- 3-14, synaptotagmin migrates at a dimer position. larger COOH-terminal fragment, which cally to the a-latrotoxin receptorin vitro This is surprising because in the inositol trisphos- migrates at a monomer phate receptor, Zwittergent 3-14 disrupts interac- position. These (11), suggestingthat this protein serves as tions between transmembrane regions totally [G. resultssuggest that tetramerizationof syn- the vesicle docking protein in the presyn- A. Mignery, C. L. Newton, B. T. Archer, T. C. aptotagmin,mediated by its NH2-terminal aptic membrane. When such a docking Sudhof, J. Biol. Chem. 265, 12679 (1990)]. Be- domain, is requiredfor formation of the is cause dimers can also be observed in SDS- complex exposedto increasedCa2+ con- PAGE, we think that two strongly bonded dimers complex with Ca2+ and phospholipids. centrations,it probablyresults in an inter- form a tetramer. Synaptotagminis the firstCa2+-binding action of the cytoplasmicarms of synap- 10. M. D. Bazzi and G. L. Nelsestuen, Biochemistry proteinthat has been identifiedin secretory totagmin with the phospholipidsof the 29, 7624 (1990). organellesof the regulatedpathway. The plasmamembrane, local 11. A. G. Petrenko et al., Nature 353, 65 (1991). causing rearrange- 12. Such lateral rearrangement of phospholipids has widespreaddistribution of at least one of ment of phospholipids(12). This may then been demonstrated for protein kinase C (where the synaptotagminisoforms on everysynap- triggerfusion via interactionwith addition- association of the protein with membranes in the tic vesicle and probablyalso every endo- al proteinsthat remainto be characterized. presence of Ca2+ leads to extensive segregation of acidic phospholipids [M. D. Bazzi and G. L. crine secretorygranule (2-4) is in agree- Nelsestuen, Biochemistry 30, 7961 (1991)] and ment with a generalrole as putative Ca2+ REFERENCESAND NOTES for various other proteins and polycations [W. receptorfor exocytosis. The precisenature Hartmann and H. J. Galla, Biochim. Biophys. Acta 1. R. Llinas, I. Z. Steinberg, K. Walton, Biophys. J. 509, 474 (1978); D. Carrier and M. Pezolet, Bio- of the interaction of synaptotagminwith 33, 289 (1981). For a review, see also L. Reichardt chemistry 25, 4167 (1986); G. B. Birrel and 0. H. phospholipidsand Ca2+ remainsto be es- and R. B. Kelly, Annu. Rev. Biochem. 52, 871 Griffith,ibid. 15, 2925 (1976); J. M. Boggs et al., tablished. Synaptotagmin, Ca2+, and (1983); G. Augustine, M. P. Charlton, S. J. Smith, ibid. 15, 5420 (1976); T. Ikeda et al., Biochim. Annu. Rev. Neurosci. 10, 633 (1987). Biophys. Acta 1026, 105 (1990)]. Furthermore, membranephospholipids probably form a 2. W. E. Mafthew, L. Tsavaler, L. F. Reichardt, J. Cell. Ca2+-dependent binding of synaptotagmin to cal- ternarycomplex or sandwich.This interde- Biol. 91, 257 (1981). modulin was reported [J. M. Trifaro, S. Fournier, pendence is similar to that observed for 3. M. S. Perin et al., Nature 343, 260 (1990); J. Biol. M. L. Novas, Neuroscience 29, 1 (1989)], which, Chem. 266, 615 (1991); M. S. Perin, N. Brose, R. however, could not be reproduced in our labora- protein kinase C, which binds Ca2+ only Jahn, T. C. Sudhof, ibid., p. 623. tories. when phospholipidsare present (10). In the 4. M. Geppert, B. T. Archer 11l,T. C. Sudhof, ibid., p. 13. Liposomes were prepared from purified phospho- absenceof Ca2+, some phospholipidbind- 13548; B. Wendland et al., Neuron 6, 993 (1991). lipids (Avanti Polar Lipids) dissolved in chloro- ing was observed(Fig. 3). This is in agree- 5. J. D. Clark et al., Cell 65, 1043 (1991). For a form:methanol, 9:1). The solvent was evaporated review, see also Y. Nishizuka, Nature 334, 661 under a stream of nitrogen and then further dried ment with our earlierobservation that the (1988). under vacuum for 30 min. Then 20 mM tris-CI (pH recombinantcytoplasmic fragment of syn- 6. The monoclonal antibody was generated by stan- 7.2) and 100 mM NaCI were added to give a final aptotagmin is capable of Ca2+-independent dard procedures [G. Kohler and C. Milstein, Na- phospholipidconcentration of 0.2 mg/mI. Phos- ture 256, 495 (1975); R. Jahn, W. Schiebler, C. pholipidswere resuspended by vigorous vortex- phospholipid binding after being subjected Ouimet, P. Greengard, Proc. Natl. Acad. Sci. ing (glass beads). Liposomes were formed by to denaturing SDS-polyacrylamide gel elec- U.S.A. 82, 4137 (1985)] with recombinant synap- ultrasonicationin a Bransonbath-sonicator for 5

1024 SCIENCE * VOL. 256 * 15 MAY 1992

This content downloaded from 132.239.70.252 on Thu, 01 Oct 2015 19:18:21 UTC All use subject to JSTOR Terms and Conditions ...... REI"ORTS

min at maximal power output. The opalescent sucrose gradients containing 0.1% of the respec- lularfiring during awake immobility (Fig. 1). emulsion was centrifuged for 20 min at 20,000g to tive detergent in the same buffer as above. For remove aggregated material. Fluorescence reso- SDS-solubilized proteins, 350 pg of total protein Sharpwaves in the stratumradiatum of the nance energy transfer assays were performed as were loaded in a 100-pl volume; for all other CAl networkreflect depolarizationof the described (10). In Fig. 2, A and B, 40 pg of purified detergents, 260 pg of total protein were loaded in apical dendritesof pyramidalcells by the synaptotagmin or synaptophysin were incubated a 300-pl volume. Gradients were centrifuged for with 10 pg of liposomes in 1 ml of 20 mM tris-CI(pH 16 hours at 4?C at 38,000 rpm in a Beckman Schaffercollaterals, which is a resultof the 7.2), 100 mM NaCI, 0.5 mM EGTA.The free Ca2+ SW41 rotor and fractionated into 24 0.5-ml frac- synchronousbursting of CA3 pyramidalcells concentration was increased by successive additions. For the experiments utilizing partially (9, 10). In conjunctionwith the stratum tions of 20 mM or 100 mM solutions of CaCI2 to trypsinized synaptotagmin, synaptic vesicles radiatumsharp waves, fast field oscillations give the free Ca2+ concentrations indicated. In Fig. were incubated with trypsin in a protein mass ratio 2C, synaptotagmin was purified from a membrane of 1:6000 before solubilization, and samples were werepresent in the CAl pyramidallayer (1, extract prepared in 1% sodium cholate instead of solubilized in CHAPS or SDS. On parallel gradi- 9, 10). The spindle-shapedoscillatory pattem Triton X-100, dialyzed against 20 mM tris-CI (pH ents, the positions of molecular size markers were consisted of 5 to 15 sinusoidwaves with 7.2), 100 mM NaCI, 1% sodium cholate, 2 mM as follows: carbonic anhydrase (29 kD), fractions phenylmethylsulfonyl fluoride, and Pepstatin A (1 2 and 3; bovine serum albumin (67 kD), fraction 4; 200-Hz intraburstfrequency. Neuronal dis- pg/ml) and combined with 60 pg of phospholipids alcohol dehydrogenase (155 kD), fraction 6; chargesmost often occurredduring the local per 100 pg of synaptotagmin resuspended in the J8-amylase (200 kD), fraction 8; apoferritin (443 fieldoscillations. Isolated pyramidal cells usu- same buffer (10% phosphatidylserine, 45% phos- kD), fractions 14 and 15. We analyzed fractions phatidylcholine, 45% phosphatidylethanolamine). by SDS-PAGE and immunoblotting, using the ally fireda singleaction potentialduring the Liposomes were formed by dialysis against Amersham enhanced chemiluminescence sys- fieldoscillations but occasionallyfired a burst cholate-free buffer. tem according to the manufacturer's directions of two to threespikes (11). The probabilityof 14. In all proteolysis experiments, the degree of pro- with polyclonal antibodies to the NH2-terminus spike bursts (complexspikes) was three to teolysis was monitored by SDS-PAGE, followed and the C2 domains of synaptotagmin as de- by fragment visualization with site-specific anti- scribed (3). eight timeshigher during the fast field oscil- bodies (3). Conditions were chosen in which all 16. We thank M. D. Bazzi for advice concerning the lationsthan duringcomparable time periods synaptotagmin was cleaved but only minor further Ca2+ equilibrium dialysis and fluorescence reso- degradation of the fragments had occurred. nance energy transfer experiments, H. Schauerte in theirabsence. 15. In the sucrose gradient centrifugations crude syn- and S. Schieback for help in some of the experi- The laminardistribution of the fast field aptic vesicles were solubilized with 1% (w/v) SDS ments, and P. R. Maycox for critical reading of the oscillationswas determined by advancinglin- or 2% (w/v) CHAPS, cholate, or Jp-octylglucoside manuscript. ear arraysof electrodes(6) perpendicularto in 75 mM tris-CI (pH 7.4), 1 mM EDTA. Solubilized proteins were loaded on 11.8 ml of 5 to 20% (w/v) 23 December 1991; accepted 9 March 1992 the CAl pyramidallayer. Amplitude maxima of the fastfield oscillations were found in the pyramidallayer (0.2 to 1 mV), and the polarityof the signalreversed in phaseabout High-Frequency Network Oscillation 100 ixm below the pyramidallayer, which suggeststhat the main currentsource of the in the Hippocampus extracellularlyrecorded fast field oscillations is the cell bodiesof pyramidalcells. Gyorgy Buzsaki,*Zsolt Horvath,Ronald Urioste, Typically,less than 15%of the recorded Jamille Hetke, Kensall Wise neuronswere active duringa single oscilla- toryepoch. When dischargesof all neurons Pyramidalcells in the CAl hippocampalregion displayed transientnetwork oscillations (200 hertz)during behavioral immobility, consummatory behaviors, and slow-wave sleep. Simultaneous,multisite recordings revealed temporaland spatial coherence of neuronal 1 Hzto10kHz activityduring population oscillations. Participatingpyramidal cells discharged at a rate 1 1 lower than the frequency of the populationoscillation, and their action potentialswere phase locked to the negative phase of the simultaneouslyrecorded oscillatoryfield potentials. In contrast, interneuronsdischarged at populationfrequency during the field 500 Hz to 10 kHz oscillations.Thus, synchronousoutput of cooperatingCAl pyramidalcells may serve to 1 1 ~`1 induce synaptic enhancement in target structuresof the hippocampus.

100 to 400 Hz Much of what is known about the physio- tions requiresthe simultaneousobservation logical function of the hippocampusis of many individualneurons in the awake 1 to 50 Hz based on in vivo and in vitro studies of animal (4). Using silicon multichannelre- 2 / sequentiallyanalyzed single neurons(1, 2). cordingarrays (5), we reporthere the phys- Althoughit has long been believedthat the iologicaldetails of a high-frequencyoscilla- computationalpower of complex neuronal tion of the hippocampalCAl neuronal networkscannot be recognizedby the prop- networkthat is a specificproduct of cellular Fig. 1. Fast field oscillationin the CAl regionof ertiesof single cells alone (3), experimental cooperativity. the dorsal hippocampus.Simultaneous record- access to the emergentproperties of coop- The data analyzedin this study were ings fromthe CAl pyramidallayer (electrode 1) eratinghippocampal neurons has been dif- recordedfrom 19 adultmale rats. Localfield and stratumradiatum (electrode 2). Uppermost ficult. Direct investigation of the time- potentials and unit activity were recorded trace of electrode 1 is wide-band recording (1 varying organizationof neuronal popula- by multichannelmicroprobes (5, 6) in the Hz to 10 kHz). Second and third traces are rat duringspontaneous behaviors and sleep digitally filtered derivatives of the wide-band trace (unit activity500 Hz to 10 kHz and fast G. Buzsaki, Z. Horvath, R. Urioste, Center for Molec- (7). The oscillatorybehavior of the record- field oscillation(100 to 400 Note simulta- ular and Behavioral Neuroscience, Rutgers University, ed cell populationsand local fieldpotentials Hz). 197 University Avenue, Newark, NJ 07102. neous occurrence of fast field oscillations,unit J. Hetke and K. Wise, Center for Integrated Sensors was determinedby the periodicmodulation discharges, and sharp wave (electrode 2). and Circuits, University of Michigan, Ann Arbor, Ml of the auto- and cross-correlograms(8). Electrode2 was 200 ,um below the pyramidal 48109. Local field potentialsin the CAl strata layer. Calibrations:0.5 mV (trace 1), 0.25 mV *To whom correspondence should be addressed. pyramidaleand radiatumwere related to cel- (traces 2 and 3), and 1.0 mV (trace 4).

SCIENCE * VOL. 256 * 15 MAY 1992 1025

This content downloaded from 132.239.70.252 on Thu, 01 Oct 2015 19:18:21 UTC All use subject to JSTOR Terms and Conditions