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Synaptic functionmodulatedbychanges in the ratio ofsynaptotagmin I and IV

J. Troy Littleton*², Thomas L. Serano²³, Gerald M. Rubin³,Barry Ganetzky* & Edwin R. Chapman§

* Laboratory of Genetics and § Department of Physiology, University of Wisconsin,

Madison, Wisconsin 53706, USA³ Department of Molecular and Cell Biology, Howard Hughes Medical Institute,

University of California, Berkeley, California 94720-3200, USA² These authors contributed equally to this work.

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Communication within the nervous system is mediated by Ca2+-triggered fusion of synaptic vesicles with the presynaptic plasmamembrane. Genetic and biochemical evidence indicates thatsynaptotagmin I may function as a Ca2+ sensor in neuronalexocytosis because it can bind Ca2+ and penetrate into lipidbilayers1±4. Chronic depolarization or seizure activity results inthe upregulation of a distinct and unusual isoform of the synap-totagmin family, synaptotagmin IV (ref. 5). We have identi®ed aDrosophila homologue of synaptotagmin IV that is enriched onsynaptic vesicles and contains an evolutionarily conserved sub-stitution of aspartate to serine that abolishes its ability to bindmembranes in response to Ca2+ in¯ux. Synaptotagmin IV formshetero-oligomers with synaptotagmin I, resulting in synaptotag-min clusters that cannot effectively penetrate lipid bilayers andare less ef®cient at coupling Ca2+ to secretion in vivo: upregulationof synaptotagmin IV, but not synaptotagmin I, decreases evokedneurotransmission. These ®ndings indicate that modulatingthe expression of synaptotagmins with different Ca2+-bindingaf®nities can lead to heteromultimers that can regulate theef®ciency of excitation±secretion coupling in vivo and representa new molecular mechanism for synaptic plasticity.

Synaptotagmins are synaptic vesicle proteins of which 12 iso-forms have been identi®ed in mammals, including many withdifferent Ca2+-binding properties6. Here we report three newsynaptotagmins from Drosophila and Caenorhabditis elegans thatare homologues of mammalian synaptotagmins IV, V and VII.Synaptotagmins contain two cytoplasmic repeats homologous tothe C2 domains found in Ca2+-dependent isoforms of proteinkinase C7. The C2A domain binds Ca2+ and anionic lipids, andtriggers the penetration of synaptotagmin I into membranes4,8,whereas the C2B domain mediates Ca2+-triggered oligomerization9.These C2 domains cooperate to form high-af®nity Ca2+-dependentcomplexes with the plasma membrane t-SNAREs syntaxin andSNAP-25 (refs 9±13).

Administration of kainic acid in rats results in clinical features ofepilepsy and status epilepticus, and causes a selective and dramaticincrease in an unusual isoform of synaptotagmin, synaptotagmin IV(ref. 5). Both the Drosophila and C. elegans synaptotagmin IVhomologue contain a conserved substitution of aspartate to serinein the third Ca2+ ligand in C2A14 (Fig. 1a). To determine whetherDrosophila synaptotagmin IV can penetrate membranes in thepresence of Ca2+, we tested the ability of immobilized recombinantcytoplasmic domains of synaptotagmins I and IV to bind to anionicliposomes in the presence and absence of Ca2+ (Fig. 1b). Fusionproteins of Drosophila synaptotagmin I containing C2A and C2ABshowed robust Ca2+-dependent phospholipid binding, whereasfusion proteins of synaptotagmin IV containing C2A or C2ABfailed to bind lipids. However, like synaptotagmin I, synaptotagminIV also interacted with the clathrin adapter AP-2, the synprintdomain from N-type Ca2+ channels, and the t-SNARE syntaxin

(Fig. 1d). The substitution of aspartate to serine is also found inmammalian synaptotagmins IV and XI (ref. 15) and phylogeneticanalyses indicate that these synaptotagmins make up a subfamily ofevolutionarily conserved synaptotagmins that fail to bind anioniclipids in response to Ca2+ (Fig. 1c).

Whole-mount embryonic in situ hybridization indicates thatsynaptotagmin IV and synaptotagmin I are expressed ubiquitouslyand speci®cally throughout the nervous system and are coexpressedin most, if not all, neurons (Fig. 1e). In order to investigate thesubcellular distribution of synaptotagmin IV, we generated antiseraagainst bacterially expressed recombinant synaptotagmin IV. Theantisera recognize a protein of relative molecular mass 55,000 thatis expressed in Drosophila head extracts but is not detected bypreimmune sera. Synaptotagmin IV, like synaptotagmin I andsynaptobrevin, is enriched in Drosophila synaptic vesicles, as assayedby subcellular fractionation of Drosophila heads (Fig. 2c). Synapto-tagmin IV is also present on synaptic vesicles immunoisolated usinganti-cysteine-string protein antibodies (data not shown), and co-sediments with other Drosophila synaptic vesicle proteins onsucrose gradients (Fig. 2d). A shift in both synaptotagmin I andIV from synaptic vesicles to presynaptic membranes in shibiremutants at the non-permissive temperature con®rms that bothsynaptotagmins are present on recycling synaptic vesicles andaccumulate in the plasma membrane during a block of endocytosis(Fig. 2d). Colocalization of synaptotagmin I and IV on the samepopulation of synaptic vesicles was con®rmed by immunoisolatingsynaptic vesicles from Drosophila head homogenates by using anti-synaptotagmin I antibodies and immunoblotting for synaptotag-min IV (Fig. 3f).

All the Ca2+ ligands are conserved in the C2B domain ofDrosophila synaptotagmin IV (Fig. 1a), indicating that Ca2+-trig-gered oligomerization may remain intact. As shown by Coomassiestaining and immunoblotting, recombinant synaptotagmins areable to form hetero-oligomeric complexes in vitro (Fig. 3a, b). Totest whether native synaptotagmin was also capable of forminghetero-oligomers, we prepared Drosophila head membranes andincubated them with either synaptotagmin I, IVor VII immobilizedrecombinant proteins. Native synaptotagmin I showed Ca2+-depen-dent binding to recombinant synaptotagmins I, IVand VII, indicat-ing that Ca2+-dependent oligomerization may be a conservedproperty of the synaptotagmin family (Fig. 3c). Co-immunopreci-pitation of synaptotagmin IV with anti-synaptotagmin I antibodiesfrom solubilized Drosophila head membranes con®rmed that thereis a speci®c interaction between the two native proteins (Fig. 3d).We next determined how the assembly of synaptotagmins into I±Iand I±IVoligomeric complexes affected their ability to interact withmembranes in the presence of Ca2+. Assembly of I±I oligomersresulted in a 45% increase in Ca2+-dependent membrane binding(Fig. 3e), whereas assembly of I±IV oligomers decreased membranebinding by 35% compared with synaptotagmin I alone, and by55% compared with I±I oligomeric complexes (P . 0:05, Student'st-test). Thus, the formation of synaptotagmin I±IV hetero-oligo-mers decreases the ability of synaptotagmin I to penetrate mem-branes, indicating that changes in the relative abundance of distinctsynaptotagmin isoforms on the same synaptic vesicle may modulatethe Ca2+ sensitivity of vesicle fusion in vivo.

To test directly the in vivo consequences of synaptotagmin IVupregulation on synaptic function, we used elav±GAL4 to driveUAS±synaptotagmin IV in a pan-neuronal manner in transgenicDrosophila. As acontrol, UAS±synaptotagmin I transgenic Drosophilawere generated and analysed in an identical manner. Immunoblotsdemonstrated a ®vefold overexpression of synaptotagmin I orsynaptotagmin IV in ¯y strains harbouring elav±GAL4 (Fig. 4d).Electrophysiological recordings at the third-instar neuromuscularjunction were performed in high-concentration extracellular Ca2+

(3 mM) for maximal stimulation of the release machinery. The peakamplitude of evoked responses from nerve stimulation was

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decreased by 21% in UAS±synaptotagmin IV; elav±GAL4 larvaecompared with controls or UAS±synaptotagmin I; elav±GAL4larvae (P . 0:05, Student's t-test) (Fig. 4a). This decrease inevoked release was of presynaptic origin, as neither muscle minia-ture excitatory junction potential (MEJP) amplitude or muscleresting membrane potential was affected. Endocytosis and vesiclerecycling also remained intact as high-frequency stimulation didnot result in further decreases in evoked responses over time

(Fig. 4b). Whereas overexpression of synaptotagmin IV decreasedevoked secretion, overexpression of either synaptotagmin I or IVresulted in decreases in MEJP frequency (Fig. 4c). Thus, bothisoforms exhibit inhibitory `clamp' actions on spontaneous fusionevents, with isoforms I and IV playing active and inhibitory roles,respectively, in evoked secretion.

Our data indicate that synaptotagmin IV can downregulate thelevel of synaptic transmission owing to loss of Ca2+-dependent

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F P R R Q I A K W H M L C D G411

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N P R R P I A Q W H T L Q V E E E V D A M L A V K K396

D. Syt IVR. Syt IVR. Syt XID. Syt IR. Syt I

b

GST

Syt I C2A

Syt IV C2A

Syt I C2AB

Syt I C2A

Syt IV C2A

0

5

10

15

*

*

GST

2 mM EGTA1mM Ca

Syt I C2AB

Syt IV C2AB

Syt IV C2AB

c

aA

DD

A L D

L P

R. Syt IO-p65bR. Syt II

D. Syt VIIR. Syt VIIM. Syt VIIIR. Syt IXD. Syt ICE Syt IA. Syt IS. Syt ICE Syt IIO-p65a

R. Syt VR. Syt VIR. Syt XCE Syt VD. Syt VCE Syt IV

R. Syt IVD. Syt IV

R. Syt XICE Syt VII

R. Syt IIIO-p65c

I

VII

IV

V

d

e

IV

GST

-Syt

IV

GST

- Sy

t I

GST

Tota

ls

Ca - + - + - + - +AP-2

N-type Ca2+

2+

2+

channel

Syntaxin

α-SNAP

Synaptobrevin

I

Pho

spho

lipid

bin

ding

(c.p

.m.×

100

0)

γ -SNAP

Figure 1 Identi®cation of the Drosophila synaptotagmin family. a, Alignment of

Drosophila synaptotagmins. Identities are shown in black, the two C2 domains in

grey, and the ®ve acidic Ca2+ ligands are boxed. Syt, synaptotagmin. b, Ca2+-

dependent phospholipid binding to immobilized recombinant synaptotagmin I or

IV. c, Dendrogram of the synaptotagmin family. D, Drosophila; R, rat; O, Discopyge

ommata; C, C. elegans; M, mouse; A, Aplysia; S, long®n squid. d, Binding of

recombinant syntaxin and synprint peptides and native AP-2 m-adaptin, a-SNAP

and g-SNAP to recombinant Drosophila synaptotagmins. Synaptotagmin I and IV

also bind inositol polyphosphates19. Interactions with SV2 have been reported for

synaptotagmin I, but this interaction is disrupted by physiological concentrations

of Mg2+, and no SV2 homologue has yet been identi®ed in Drosophila. e, Whole-

mount in situ hybridization to stage 16 (synaptotagmin IV) or 17 (synaptotagmin I)

Drosophila embryos with digoxigenin-labelled probes.


phospholipid binding activity, coupled with its ability to assembleinto oligomers with synaptotagmin I, thereby allowing the synapseto adapt a new state of activity. Upregulation of synaptotagmin IVin response to seizure activity may have evolved as an adaptivemechanism to decrease neural activity. Although we cannot excludethe possibility that increased synaptotagmin IV expression maydownregulate release by an unknown mechanism, the only bio-chemical difference we found between synaptotagmin I and IV istheir ability to bind phospholipids in a Ca2+-dependent manner.The large number of different synaptotagmin isoforms with uniqueCa2+-binding af®nities, and the ®nding that mammalian synapto-

tagmin isoforms can also assemble into heteromultimers16,17, pro-vide the potential for a large combinatorial `library' of Ca2+ sensorsthat assemble in vivo to modulate synaptic strength. M. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Methods

Binding assays and production of recombinant proteins and antibodies.

Complementary DNA encoding the cytoplasmic domains of Drosophila

synaptotagmins I (C2A, residues 144±317; C2AB, residues 144±474), IV

(C2A, residues 132±323; C2AB, residues 132±474) and VII (C2AB, residues

28±417) were expressed as glutathione S-transferase fusion proteins as

described13. Thrombin was used to cleave synaptotagmin IV from GST and

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NATURE | VOL 400 | 19 AUGUST 1999 | www.nature.com 759

Syntaxin

Tota

lHea

vy m

embra

nes

Light

membra

nes

Cytoso

l

Synaptobrevin

Synaptotagmin I

Synaptotagmin IV

SNAP-25

Coomassie

anti-SYT I

anti-SYT IV

SYT I

SYT IVb ca

UAS-SYT IV

10278

49.5

3428

Contro

l

Syntaxin

Synaptobrevin

Synaptotagmin I

Synaptotagmin I

Synaptotagmin IV

Gradient fraction

1 2 3 4 5 6 7 8 9 10

Shibire

CSP

TopBottom

ShibireSynaptotagmin IV

d

Mr (K)

Figure 2 Synaptotagmin IV is a synaptic vesicle protein. a, Western blot of

dissected imaginal discs from control or UAS±synaptotagmin IV; sevenless±

GAL4 larva. The synaptotagmin IV antisera speci®cally recognize a protein of

relative molecular mass 55,000 (Mr 55K) from the synaptotagmin IV gene under

the control of the UAS promoter. b, Top, Coomassie gel of synaptotagmins I and

IV. Bottom, immunoblots probed with isoform-speci®c antibodies. c, Synapto-

tagmin IV is concentrated in light membrane fractions enriched in synaptic

vesicles. d, Sucrose gradient velocity sedimentation fractions of Drosophila head

membranes from CS or heat-shocked shibire adult ¯ies were probed for antigens

as shown. Plasma membrane proteins such as syntaxin and the sodium pump

migrate to the bottom of the gradient in the last two fractions. In contrast, synaptic

vesicle proteins migrate in fractions 3±6. In shibire mutants, synaptic vesicle

proteins accumulate in the plasma membrane.

Totals

GST-Syt

I C2A

B

GST-Syt

IV C

2AB

- + - + - +

Syt IV C2AB

Syt I C2AB

Syt I (native)

Totals

GSTGST-S

yt IV

C2A

B

- + - + - +

Totals

GSTGST-S

yt I C

2AB

Ca2+

Ca2+

Ca2+

- + - + - +

0

1,000

2,000

3,000

4,000

(c.p

.m.)

3 H-p

hosp

holip

id b

indi

ng

Syt I + 1X Syt I C2AB

Syt I + 5X Syt I C2AB

Syt I + 1X Syt IV C2AB

Syt I + 5X Syt IV C2AB

Syt I + EGTA

Syt I + Ca 2+

a

b

c

e

Totals

Beads

Contro

l Ab

- Bea

ds

Synaptobrevin

Syt I A

b - B

eads

Synaptotagmin IV

d

f

- + GST-S

yt VII C

2AB

Totals

Beads

Syt I A

b - B

eads

Synaptotagmin IV

Synaptobrevin

Synaptotagmin I

Figure 3 Hetero-oligomerization of Drosophila synaptotagmins I and IV

decreases membrane binding. Binding was detected by Coomassie staining

(a, b) or by immunoblotting using an anti-synaptotagmin I luminal domain

antibody (c). a, Binding of 1 mM recombinant synaptotagmin (Syt) IV cytoplasmic

domain (C2AB) to 30 mg of immobilized recombinant synaptotagmin I C2AB in

100 ml. b, Binding of 1 mM recombinant synaptotagmin I cytoplasmic domain to

30 mg immobilized recombinant synaptotagmin IV C2AB in 100 ml. c, Binding of

native synaptotagmin I from Drosophila head detergent extracts to 30 mg

recombinant immobilized synaptotagmins I, IVand VII.d, Co-immunoprecipitation

of synaptotagmin IVwith synaptotagmin I from Triton X-100 solubilized Drosophila

head membranes incubated in 1mM Ca2+ using anti-synaptotagmin I antibodies

cross-linked to protein G beads. e, Phospholipid binding to synaptotagmin I±IV

hetero-oligomers. f, Synaptotagmin IV is found on synaptotagmin I immuno-

isolated vesicles. Similar results are seen with mammalian synaptotagmin IV (refs

17, 20).


the cleaved material was separated by SDS±PAGE and used to immunize

rabbits according to standard procedures, using the University of Wisconsin

polyclonal antibody facility. For testing binding of native synaptotagmin I to

recombinant synaptotagmins, detergent extracts from Drosophila head mem-

branes were prepared13 and 2 mg of extract was incubated with 30 mg recombi-

nant protein, and 7.5% of total and 17% of bound material was loaded onto

gels. Synaptotagmin homo- and hetero-oligomers were assembled by adding

either equimolar or a ®vefold molar excess of recombinant synaptotagmin I or

IV to 15 mg immobilized synaptotagmin I for 2 h in 1 mM Ca2+. Unbound

soluble synaptotagmin was removed by washing three times before samples

were assayed for phospholipid binding activity. Velocity sedimentation was

carried out using 10±30% sucrose gradients centrifuged for 1 h at 50 r.p.m. in a

VTi65 rotor. Immunoisolation of synaptic vesicles and immunoprecipitation

from Drosophila head membranes was as described18.

Generation of transgenic Drosophila. A 2-kilobase (kb) fragment encom-

passing the synaptotagmin I open reading frame (ORF) and a 3.1-kb fragment

spanning the synaptotagmin IV ORF (obtained from the Drosophila Berkeley

Genome Project) were subcloned into PUAST vectors, and transgenic

Drosophila were generated by germline transformation.

Electrophysiological analysis. Electrophysiological recordings were per-

formed on male third-instar non-Tb larva from crosses of UAS±synaptotagmin

I or IV virgins to elav±GAL4; T(2:3) CyO±TM6Tb males in 3 mM Ca2+-HL3 as

described2 using an Axoclamp 2B.

Phospholipid binding assays. Phospholipid assays were performed with

25%PS/75%PC using 15 mg recombinant protein as described4.

Received 28 May; accepted 30 June 1999.

1. Littleton, J. T., Stern, M., Schulze, K., Perin, M. & Bellen, H. J. Mutational analysis of Drosophila

synaptotagmin demonstrates its essential role in Ca2+-activated neurotransmitter release. Cell 74,

1125±1134 (1993).2. Littleton, J. T., Stern, M., Perin, M. & Bellen, H. J. Calcium dependence of neurotransmitter release

and rate of spontaneous vesicle fusions are altered in Drosophila synaptotagmin mutants. Proc. Natl

Acad. Sci. USA 91, 10888±10892 (1994).

3. Geppert, M. et al. Synaptotagmin I: a major Ca2+ sensor for transmitter release at a central synapse.

Cell 79, 717±727 (1994).

4. Chapman, E. & Davis, A. Direct interaction of a Ca2+-binding loop of synaptotagmin with lipidbilayers. J. Biol. Chem. 273, 13995±14001 (1998).

5. Vician, L. et al. Synaptotagmin IV is an immediate early gene induced by depolarization in PC12 cells

and in brain. Proc. Natl Acad. Sci. USA 92, 2164±2168 (1995).

6. Sudhof, T. & Rizo, J. Synaptotagmins: C2-domain proteins that regulate membrane traf®c. Neuron 17,

379±388 (1996).7. Perin, M. S., Fried, V. A., Mignery, G. A., Jahn, R. & SuÈdhof, T. C. Phospholipid binding by a synaptic

vesicle protein homologous to the regulatory region of protein kinase C. Nature 345, 260±263 (1990).

8. Brose, N., Petrenko, A. G., Sudhof, T. C. & Jahn, R. Synaptotagmin: a Ca2+ sensor on the synaptic

vesicle surface. Science 256, 1021±1025 (1992).

9. Chapman, E. R., An, S., Edwardson, J. M. & Jahn, R. A novel function for the second C2 domain ofsynaptotagmin: Ca2+-triggered dimerization. J. Biol. Chem. 271, 5844±5849 (1996).

10. Chapman, E. R., Hanson, P. I., An, S. & Jahn, R. Ca2+ regulates the interaction between synaptotagmin

and syntaxin 1. J. Biol. Chem. 270, 23667±23671 (1995).

11. Schiavo, G., Stenbeck, G., Rothman, J. & Sollner, T. Binding of the synaptic vesicle v-SNARE,

synaptotagmin, to the plasma membrane t-SNARE, SNAP-25, can explain docked vesicles atneurotoxin-treated synapses. Proc. Natl Acad. Sci. USA 94, 997±1001 (1997).

12. Li, C. et al. Ca2+-dependent and -independent activities of neural and non-neural synaptotagmins.

Nature 375, 594±599 (1995).

13. Littleton, J. T. et al. Temperature-sensitive paralytic mutations demonstrate that synaptic exocytosis

requires SNARE complex assembly and disassembly. Neuron 21, 401±413 (1998).14. Sutton, R., Davletov, B., Berghuis, A., Sudhof, T. & Sprang, S. Structure of the ®rst C2 domain of

synaptotagmin I: a novel Ca2+/phospholipid-binding fold. Cell 80, 929±938 (1995).

15. von Poser, C., Ichtchenko, K., Shao, X., Rizo, J. & Sudhof, T. The evolutionary pressure to inactivate:

A subclass of synaptotagmins with an amino acid substitution that abolishes Ca2+ binding. J. Biol.

Chem. 272, 14314±14319 (1997).16. Chapman, E., Desai, R., Davis, A. & Tornehl, C. Delination of the oligomerization, AP-2 binding, and

synprint binding region of the C2B domain of synaptotagmin. J. Biol. Chem. 273, 32966±32973

(1998).

17. Osborne, S. L., Herreros, J., Bastiaens, P. I. & Schiavo, G. Calcium-dependent oligomerization of

synaptotagmins I and II. J. Biol. Chem. 274, 59±66 (1999).18. van de Goor, J., Ramaswami, M. & Kelly, R. Redistribution of synaptic vesicles and their proteins

in temperature-sensitive shibire(ts1) mutant Drosophila. Proc. Natl Acad. Sci. USA 92, 5739±5743

(1995).

19. Fukuda, M., Kojima, T., Aruga, J., Niinobe, M. & Mikoshiba, K. Functional diversity of C2 domains of

synaptotagmin family. J. Biol. Chem. 270, 26523±26527 (1995).20. Ferguson, G. D., Thomas, D. M., Elferink, L. A. & Herschman, H. R. Synthesis degradation, and

subcellular localization of synaptotagmin IV, a neuronal immediate early gene product. J. Neurochem.

72, 1821±1831 (1999).

Acknowledgements. We thank R. Kelly, S. Benzer, L. Brodin, C. Goodman, W. Caterall, H. Jackle andR. Jahn for antibodies and reagents, and J. Pendleton for help with sequencing. This work was supportedby grants from the NIH and the Howard Hughes Medical Institute. T.L.S. is a Jane Cof®n Childspostdoctoral fellow. J.T.L. is a Merck fellow of the Helen Hay Whitney Foundation.

Correspondence and requests for materials should be addressed to J.T.L. (e-mail: [email protected]).

letters to nature

760 NATURE | VOL 400 | 19 AUGUST 1999 | www.nature.com

10 mV

50 ms

Syt I - UAS; elav-GAL4 Syt IV - UAS; elav -GAL4Syt I - UAS Syt IV - UAS

a

b

10 mV

100 ms

Syt IV - UAS; elav-GAL4 Syt I - UAS; elav -GAL4

Syt IV - UAS; elav-GAL4

Syt I - UAS

Syt IV - UAS

0

1

2

3

4c

**

ME

JP F

requ

ency

(s

–1)

Syt IV

- UAS; e

lav-G

AL4

white

Syt I -

UAS; elav

-GAL4

anti-Syt I

anti-Syt IV

d

anti-Syt I

anti-Syt IV

Whole larva

Larval fillets

Figure 4 Electrophysiological analysis of transgenic Drosophila overexpressing

synaptotagmin IV or synaptotagmin I. a, Transgenic larvae containing either

synaptotagmin I±UAS or synaptotagmin IV±UAS alone or with elav±GAL4 to drive

neuronal overexpression were recorded in 3mM Ca2+ HL3 solution. Peak

amplitude of the excitatory junctional potential and the resting membrane

potential from muscle ®bre 6 were as follows for each genotype: synaptotagmin

I±UAS (39:3 6 2:1, 56 6 1:8, n � 8); synaptotagmin I±UAS; elav±GAL4 (40:6 6 2:1,

60 6 1:9. n � 17); synaptotagmin IV±UAS (41:4 6 2:1, 60 6 2, n � 14);

synaptotagmin IV±UAS; elav±GAL4 (32:9 6 2:6, 58 6 1:7, n � 19). b, Stimulation

(10Hz) of a neuromuscular junction from synaptotagmin IV±UAS; elav±GAL4

larvae showed no depletion over time. c, MEJP frequency was decreased by

overexpression of either synaptotagmin I or IV (P , 0:05, Student's t-test). d, Five

larvae of the indicated genotypes were homogenized in sample buffer. Immuno-

blots with anti-synaptotagmin I or anti-synaptotagmin IV antisera showed a

speci®c ®vefold upregulation of the corresponding protein in elav±GAL4 larva

containing the UAS transgenes compared to controls. Similar levels of over-

expression were observed in immunoblots of larval neuromuscular junction

®llets, as used for electrophysiology (bottom).

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