Journal of Science iso fnwyfre eilsfo h lsammrn.In membrane. mediates plasma that the sensitive from machinery vesicles Kelly, temperature formed and the newly Roos of 2004; of al., part fission et is Marie 2004; al., 1998). et Koh gene 2007; GTPase al., the the et is by complex encoded protein dynamin, scaffolding 2009; the Ryan, with interactions and (Dittman understood 2010). al., poorly the et Pechstein is of function identification precise the their the , in Although in scaffolding the of made membrane. of partners been binding presynaptic regulation has the progress in significant at recruit to proteins, platform network implicated F-bar a as cytoskeletal to act effectors and binding 2010) al., via endocytic et (Henne sites they 2 endocytic and that FCHo1 shown at have localized loss cells are their non-neuronal and in in scaffold Experiments defects 2007). molecular similar a very as to leads act 15, proteins clone Both substrate pathway Eps15. receptor factor growth epidermal and eprtr,ms V uewt h rsnpi membrane, presynaptic the with fuse SVs most temperature, fetbl ebaertivli synapses in A retrieval membrane bulk Dap160/intersectin affect of domains dynamin-binding The Article Research oeue,sc sdnmnascae rti 6 kDa, 160 protein dynamin-associated as in scaffolding Dap160, such Camilli, large by De these coordinated molecules, executing is and proteins events effector (Saheki trafficking endocytic membrane (PAZ) of action zone at The operating 2012). periactive recycling synaptic (SV) vesicle two the synaptic the for are pathways endocytosis major bulk and endocytosis -mediated Introduction the to dynamin of targeting activity. the high-frequency in during Dap160 retrieval membrane of vesicle role synaptic important stimulation, bulk words: dye the high-rate suppress Key the of during reveal to number of (EJPs) required the data uptake potentials in is The in increase junction it reduction EJPs. an excitatory where a observe miniature evoked zone, not by in large periactive do accompanied depression we aberrantly is a However, by This invaginations. note accompanied membrane stimulation. also and during role We vesicles key bouton intermediates. that large a the clathrin-coated of show plays in accumulation but we an dispersed junction Here and becomes neuromuscular understood. FM1-43 the it well of and development not zone, the is recycling. periactive affect domains vesicle not interaction does and synaptic domains protein endocytosis in SH3 affects different dynamin-binding and the the proteins lacking of synaptic Dap160 role several functional with interacts The (Dap160)/intersectin development. kDa 160 protein Dynamin-associated Summary USA 10032, NY York, New Center, Medical University Columbia Disease, and Biology § Motor for ` Center address: *Present 3 2 1 o:10.1242/jcs.118968 doi: 1021–1031 126, Science Cell of Journal 2012 December 6 Accepted ae .Schulze L. Karen ˚ rsn drs:Dp fMlclr ellradDvlpetlBooy aeUiest,26WinyAeu,NwHvn T051 USA 06511, CT Haven, New Avenue, Whitney 266 University, Yale Biology, Developmental and Cellular Molecular, of Dept address: Present uhrfrcrepnec ( correspondence for Author eateto oeua n ua eeisadHM,NI alrCleeo eiie oso,T 73,USA 77030, TX Houston, Medicine, of College Baylor USA NRI, 77030, HHMI, TX and Houston, Genetics Medicine, Human of and College Molecular Baylor va of NRI, Eulers Department Biology, von Developmental Institutet, in Karolinska Program DBRM, Neuroscience, of Department aM .Winther E. M. sa 03 ulse yTeCmayo ilgssLtd Biologists of Company The by Published 2013. n ftekyedctcefco rtisipiae in implicated proteins effector endocytic key the of One cfodn oeue,Poenmgain H oan ermsua junction, Neuromuscular domain, SH3 migration, Protein molecules, Scaffolding 3 [email protected] uoJ Bellen J. Hugo , 1 e Jiao Wei , shi temmainotoo sintersectin), is ortholog mammalian (the ts1 uat eta h restrictive the at kept mutants dap160 shibire 1, ,Og Vorontsova Olga *, ) uat akn yai-neatn oan olne cuuaednmnpoel tthe at properly dynamin accumulate longer no domains dynamin-interacting lacking mutants ( shi 2,3 nfis(Evergren flies in ) nvivo in n lgShupliakov Oleg and Khe al., et (Koh 1 ahy .Rees A. Kathryn , g3 7 7Sokom Sweden Stockholm, 77 171 3, ¨g i o xli o tflildti function. may this fulfilled Dap160 they it but that how PAZ, and explain al., the suggestion not at et proteins did the endocytic Marie endophilin, of to 2004; function the led al., coordinate several findings dynamin, et of These (Koh of reported 2004). levels including also amplitude in was decrease synaptojanin larger proteins, 50% and to endocytic Up activity events. spontaneous spontaneous of frequency eid y paewssgiiatyrdcd(ai ta. 2004). al., et the (Marie reduced However, significantly was uptake dye period, rnmsin(o ta. 04.Smlry saso synaptic short-term of no revealed assays loading in Similarly, dye defects FM 2004). with al., as endocytosis vesicle et such Low synaptic in (Koh activity, impairments 2004). revealed stimulation, transmission high-frequency Hz al., 10 of of et minutes 10 conditions Marie 1998). 2004; Only in Kelly, EJPs al., normal mutants. near and in et results stimulation (Koh nerve Roos frequency dynamin 2004; the of than severe loss al., less much are that et (NMJs) of junctions neuromuscular loss Marie complete Koh the 2004; 2004; Surprisingly, (Broadie, al., function dynamin et of aspects for important In 1998). Kelly, numerous Drosophila and (Roos Dap160/intersectin of of modules domain 2012). accumulation Camilli, De and and of (Ferguson block pits a recycling coated in constricted results vesicle also with genes dynamin synaptic intermediates mammalian deletion three Genetic endocytic all accumulate. of vacuoles and large and blocked collars dynamin is endocytosis but 1,§ neatoswt yai nov eea r oooy(SH3) homology Src several involve dynamin with Interactions dap160 ev emnl hsbnighsbe rpsdt be to proposed been has binding this terminals nerve 1 ogWyKoh Tong-Wey , dap160 Drosophila uat,atog fe 0mnt labeling 10-minute a after although mutants, ulmtnsdslydtoodhigher twofold displayed mutants null 2, dap160 ` ln Sopova Elena , asspeoye in phenotypes causes Drosophila 1 dap160 , 1021 Journal of Cell Science ate,dnmn trs n uigsnpi ciiyfollowing activity synaptic binding during its and and rest Dap160 at of activity dynamin, localization synaptic partner, the during PAZ investigated the first to We relocates vesicles synaptic and of rest pool at distal the in accumulates Dap160 1022 Results clathrin- SV for bulk critical not controlling is in but endocytosis. role NMJ, mediated the essential at an trafficking between plays membrane and interaction proteins activity The PAZ. two synaptic the the during at dynamin zone concentrates its the periactive it from study that the relocates to to lacking Dap160 pool that order vesicle proteins show in experiments Dap160 Our function. mutant in express the modules used dynamin-interacting to We PAZ. background the null at functions dynamin coordinate ntepeetsuyw netgt o a10may Dap160 how investigate we study present the In ora fCl cec 2 (4) 126 Science Cell of Journal dap160 xouet 0m K mM 60 to exposure crls aebe bevdpeiul n r huh to and Roos thought 2004; are al., et and (Marie Such previously zones 1C). Dap160-ir periactive (Fig. of observed synaptic spots The been outline Brp level significantly 1D). have around (Fig. circles the ‘circles’ Brp-ir clear synapses in while and stimulated accumulated in Dap160-ir S1A), decreased between Fig. colocalization material (supplementary eanducagdbt trs n pnhg K high punctae upon individual and of rest size at The 2006). both position al., unchanged et the remained (Wagh indicated T-bar which the spots, of in The accumulated 1C,D). labeling (Fig. (Brp) Brp the zone, Bruchpilot and component, active Dap160 T-bar against the presynaptic antibodies within with distribution NMJs double-stained their we examine images To confocal within 1A). in proteins evident (Fig. was the stimulation of under redistribution under terminals A nerve colocalized 1B). strongly (Fig. conditions proteins both Both 1A). (Fig. microscopy atce (particles/ particles 2 e)a etaduo ihK high upon and (Brp, rest Bruchpilot at and red) (green) Dap160 double- for boutons labeled of sections middle optical showing ( (PCC). Pearson’s coefficient by correlation and measured rest as at stimulation, Dap160 following and dynamin of colocalization otebre ftepoia olo vesicles. of pool proximal the ( of border correspond the outlines to Dashed (J). dynamin-ir and Dap160-ir (G) of images High-magnification F particle. (G,J) in gold Asterisk silver-enhanced (I). a dynamin-ir indicates and (F) Dap160-ir of distribution the showing NMJs immunogold-labeled Low from (F,I) micrographs rest. at electron vesicles magnification synaptic of pool distal the Syt. with overlapping ( not is in Dap160 area an which indicate Arrows red). and (Syt, (green) Dap160 for double-labeled NMJ upon ( decreases stimulation. Brp and Dap160 between observed. is ( but colocalization anti-Brp, of by degree labeled some areas the surrounds often yatcvsce a rpsidct en+s.e.m. + mean indicate *** graphs sv, Bar ; vesicle. m, synaptic and T-bar; (H) T, Dap160 (K). for dynamin vesicles synaptic of pools pnepsr ohg K and high rest to at exposure red) upon (Dyn, dynamin and (green) Dap160 ( rcplti oto ( and control dynamin in Dap160, Bruchpilot of Localization 1. Fig. nue ysiuain ( stimulation. boutons by in induced areas unlabeled indicate Arrowheads H F D A + m , uniiainsosta h colocalization the that shows Quantification ) ofcliae fNJ obelbldfor double-labeled NMJs of images Confocal ) , G K ACE;0.5 (A,C,E); m hg K (high P , , a rpssoigdniiso gold of densities showing graphs Bar ) I , J .0 sn Student’s using 0.001 a10addnmnaecnetae to concentrated are dynamin and Dap160 ) + E o 0mntsuigconfocal using minutes 10 for ) ofclmcocp mg fan of image microscopy Confocal ) m m m FI;0.1 (F,I); m 2 ntedsa n proximal and distal the in ) w + B 1118 uniiaino the of Quantification ) o 0mnts(stim). minutes 10 for + tmlto.Dap160-ir stimulation. hr ntrNMJs. instar third ) t ts.Saebars: Scale -test. C ofclimages Confocal ) m (G,J). m + stimulation Journal of Cell Science olrdpt ttepam ebae ossetwt oeof role 2C,D). a (Fig. with endocytosis consistent constricted in membrane, dynamin containing and plasma Dap160 the regions at membrane pits collared the with associated oaiaino a10a etuigsiuae emission Dap160 that Fig. stimulated show NMJs material using through images (supplementary rest STED Serial microscopy at S1B–D). (STED) Dap160 synaptic depletion of undefined localization an both that stimulation. from upon suggests PAZ relocate This the to dynamin 1999). compartment Kelly, and and Dap160 Roos 1998; Kelly, uat xoe otersrcietmeaue(29 temperature restrictive the to exposed mutants K high NMJs fixed to we exposure activity, after synaptic during change dynamin SVs. of partner localize pool distal the experiments to dynamin Brp our are partner binding and surroundings and its and immediate CSP Dap160 immunolabeling, its of for and patterns T-bar accessible Brp labeling the (supplementary of that The T-bar demonstrate C-terminus S2B,C). the from Fig. the emerging material against filaments raised Fig. the material labeled antibody (supplementary SVs The of distributed S2D–F). pool evenly whole were the CSP labeling throughout As particles our S2B–F). gold Fig. antibodies material the and with structures (supplementary 1994), expected with 2006) al., al., labeling et and NMJs et (Zinsmaier (Wagh CSP Brp to the vesicles protein, string stained accessible cysteine these against we are that approach T-bar immunogold confirm the material To supplementary surrounding 1G,H,J,K; S2A). (Fig. pedestal presynaptic labeled Fig. T-bar the not the surrounding of were SVs immediately borders of T-bar SVs the pool The from the 1F–K). the away in (Fig. nm at found 100 were labeling than dynamin were the more and of particles subcellular Dap160 visualization gold level. the improve EM The to clarify dynamin. enhanced and further silver Dap160 to of technique localization immunogold the 2010). al., et (Pechstein intravesicular pool the SV in the This localizes within rest 1E). matrix at (Fig. Dap160 Dap160 seen that clearly suggests if are further are synaptotagmin determine punctae with Dap160 and to only colocalize, and labeled completely 1993), Synaptotagmin not do marker. but al., overlapping SV largely et a Littleton with colocalizes 1996; (Estes protein al., SV-associated a et synaptotagmin, the for then to NMJs We close S1C,D). labeled or Fig. at material (supplementary located membrane bouton plasma the of areas unlabeled surrounds gemn ihimnfursec aa ohi oto and control In in 2C,D). both (Fig. data, 1989) shi Ikeda, machinery immunofluorescence and recycling with (Koenig vesicle PAZ agreement the synaptic at the ‘locked’ which is in minutes, 10 n irtdt h eiciezn i nidpnetpathway independent the an were In via that release zone 2A,B). particles periactive (Fig. suggesting during the gold to SVs membrane, migrated to the and numerous bound plasma not of Interestingly, were contained the proteins majority 2H,I). still at (Fig. the concentrated terminals region vesicles, control this synaptic gold many and in Dap160 of although in Quantification labeling increase significant 2A–I). dynamin a (Fig. revealed PAZ densities particle the in concentrated oaie ntePZa h Mlvlw lostained also we level EM the are proteins at the PAZ that the verify plasma To the in 2A,B). to localized (Fig. close PAZs labeling in found membrane and technique immunogold enx sdeeto irsoy(M ncnucinwith conjunction in (EM) microscopy electron used next We subcellular the studied we compartment this define To odtriehwtelclzto fDp6 n t binding its and Dap160 of localization the how determine To ts1 rsnpi emnl,Dp6 n yai eefound were dynamin and Dap160 terminals, presynaptic shi ts1 uat lotalgl atce were particles gold all almost mutants + o 0mntsadsanduigthe using stained and minutes 10 for ˚ )for C) shi a10codntsbl noyoi nsnpe 1023 synapses in endocytosis bulk coordinates Dap160 ts1 aiu H oancmiain fDp6 hwdthat showed and SH3AB with Dap160 4A). (Fig. experiments module of binding dynamin pull-down combinations key the is GST domain SH3B domains SH3B SH3 4A). Kelly and (Fig. and SH3A various the Roos dynamin only of that bind observations 1998) this, Kelly, earlier accomplish and the To (Roos confirmed modules. first interacting we dynamin the lacking ed oaseii itreigo yai rmtePZduring PAZ the from dynamin of of stimulation. mistargeting deletion specific Hence, a to S3E). leads Fig. material (supplementary h A,wie7%wr ntePZi tmltdcontrols, stimulated in PAZ stimulated in the were in in particles were 90% gold 70% of and while 10% PAZ, only synapses the control resting In ete sesddnmndsrbto in distribution dap160 dynamin PAZ assessed the then from We of Eps15 mislocalization not the but in dynamin results Dap160 of Deletion netgt h ucinlrl fdnmntreigt the generated to we targeting dynamin Dap160, a of by indicating To 2004). role boutons, zone al., et functional satellite periactive Marie of 2004; the al., appearance investigate et as (Koh the dynamin, defect including to developmental proteins, as endocytic several well of levels the of deletion domains Genetic SH3 a in dynamin-interacting PAZ the the lacking from mislocalized is Dynamin ntePZcmae osiuae oto Fg Q.When 3Q). (Fig. control stimulated to stimulated compared to compared PAZ immunolabeling the dynamin in of in membrane reduction relative presynaptic a showed the particles along at distributed concentrated is dap160 not and is PAZ dynamin Immunogold the that S3B,C). confirmed Fig. experiments EM material supplementary 3E,F,J; (Fig. eoaefo h itlvscepo eint h A during PAZ the to region pool dynamin vesicle activity. partner distal synaptic binding the its from and relocate Dap160 that show experiments eraei yai odpril est sdtce tthe at detected is density particle gold Fig. rest dynamin in material in EM-level, at supplementary decrease the control 3A–C,G,I; At (Fig. adaptor S3A,C). rest endocytic to the AP2 at and pool Eps15 compared with vesicle complex, colocalized the largely with is when it associated and is Dynamin mutants dramatically 3A,G). (Fig. not in EM is distribution immunogold changed dynamin that and show Immunofluorescence experiments activity. synaptic Fg DH.Hwvr p1 ssillclzdt endocytic to and control localized NMJs in as still control AP2 is stimulated with colocalizes in Eps15 and punctae unlike However, of PAZ, 3D,H). spots in (Fig. the accumulated control longer at no is fluorescence versus and terminal labeling the throughout Eps15 for S3D). observed Fig. 3K–M,P; material (Fig. is (supplementary control decrease to no compared PAZ Msa etaddrn yatcatvt ncnrland control in activity synaptic during of sections and ultrathin serial rest shi from 3D at in NMJs Dap160 for sites release itiuino odprilsfrEs5wsntsignificantly not was Eps15 for control, stimulated particles comparing different gold of distribution n 72.7 (from particles threefold gold reduced dynamin-immunoreactive was with associated PAZ the 5 nstimulated In ete eosrce muoodpril oaiainat localization immunogold-particle reconstructed then We ts1 5 mean 15; uat(i.2–;splmnaymtra i.S2G–I). Fig. material supplementary 2E–G; (Fig. mutant D uatNJbuos(i.3,) uniiaino gold of Quantification 3N,O). (Fig. boutons NMJ mutant 1 /Df(2L)bur-K1 6 SD, dap160 dap160 P , shi .01 Student’s 0.0001, ulmtn Ms yai sdispersed is dynamin NMJs, mutant null Khe l,20) trs n during and rest at 2004), al., et (Koh shi ts1 eut nadces faot5%of 50% about of decrease a in results h ecnaeo noyi isat pits endocytic of percentage the , ts1 uat.Tkntgte,these together, Taken mutants. dap160 6 24.9%; dap160 ulmtnsa et a rest, at mutants null n dap160 5 t ts) h relative The -test). 9t 23.7 to 19 P and , dap160 dap160 .5,whereas 0.05), shi ulmutants, null shi ts1 6 ts1 mutants mutant animals dap160 26.3%; NMJs Journal of Cell Science DAudrUScnrlsre sapstv control positive a mutated as and served full-length control The UAS 4B). (Fig. under cDNA ( domains ( domain Two ytmadanua-pcfcGL rvr(elav-GAL4 GAL4/UAS driver the GAL4 using neural-specific a them and expressed system and background null HADdslydsrne idn hnS3 alone, the modules. in SH3B domain transgenics SH3 than created the then binding among We cooperativity stronger suggesting displayed SH3A-D 1024 ibeaut,wihwr etl n bet l.Fo here From fly. to mutated able respective and fertile were onwards, which adults, viable ece h ehlt of lethality the rescued on yai rln-ihdmi PD osnot it full-length GST- whereas and transgenics, does wild-type mutant in that our Dap160 (PRD) of precipitated extracts successfully confirmed domains head domain SH3B the and from then SH3A proline-rich lacking Dap160 immunoprecipitate We dynamin bound background. mutant dap160 D ora fCl cec 2 (4) 126 Science Cell of Journal D D )adteohrlcigbt HAadSH3B and SH3A both lacking other the and B) B.Aiasta xrse h full-length the expressed that Animals AB). B rngnswr eeae,oelcigteSH3B the lacking one generated, were transgenes and D dap160 AB eest nml htepesthe express that animals to refers dap160 rngnsi the in transgenes D 1 /Df(2L)bur-K1 dap160 dap160 D 1 /Df(2L)bur-K1 dap160 uat to mutants transgenes dap160 C155 null ). tescniin teeae eprtr (34 temperature elevated at conditions stress eprtr-estv ulmtn sprlzd locomotion a paralyzed, is in defect mutant null temperature-sensitive ciiywsosre in observed was activity D ececnrl splmnaymtra i.SA.Aweak of A case in S4A). 25 at detectable performed Fig. assay still was material band (supplementary Dap160 controls rescue i o eelsgiiatdfeecsi yai xrsinin expression dynamin of the in differences in number immunofluorescence significant NMJs in addition, reveal changes In not or 4C–F). did (Fig. architecture boutons synaptic by satellite in terminals defects nerve display not any did to modules, dynamin-interacting dynamin lacks which the of However, mutant, development. synaptic delivery for important that is Dap160 possible is It of architecture synaptic the 4H). (Fig. defect synaptic a suggesting aeilFg 4–) hs neatoso a10involving Dap160 of interactions Thus, S4B–E). Fig. material AB ale tde aervae eeedvlpetldfcsin defects developmental severe revealed have studies Earlier hr ntrlra.Hwvr ag eraei locomotor in decrease large a However, larvae. instar third D AB 0.1 * s.e.m. + upon mean *** zone indicate periactive graphs the Bar to stimulation. particles gold of serial ( distribution in case. studied each were for zones sections active as Eight shown spheres. are black are particles zones immunogold active Silver-enhanced gray, violet. depicted in is Dap160-ir membrane of Plasma localization synapses. subcellular the of ( reconstructions intermediates. endocytic indicate Arrows terminals. cuuae ttepam ebaei stimulated in membrane pits plasma the endocytic at invaginated accumulated with associated are zone. dynamin-ir periactive the at (Dyn) ( dynamin-ir and Dap160-ir of fnretriaso oto hr ntrlra oto after control larvae K instar high third to control exposure of terminals nerve of zone. and periactive Dap160 the of to redistribution dynamin causes Stimulation 2. Fig. C D infcnl ifrn rmcnrl a observed, was controls from different significantly , D P AB m ihmgiiainiae hwn htDp6-rand Dap160-ir that showing images High-magnification ) , m. .0 sn NV,Tkysps et cl bars: Scale test. post Tukey’s ANOVA, using 0.001 uatcmae ocnrl(supplementary control to compared mutant ˚ i o eelsgiiatmtrdfcsin defects motor significant reveal not did C dap160 dap160 + o 0mntssoiga accumulation an showing minutes 10 for H ulmtns(i.4) Under 4G). (Fig. mutants null , ulmtns(o ta. 2004). al., et (Koh mutants null I a rpssoigterelative the showing graphs Bar ) ( A , B lcrnmicrographs Electron ) D ˚ ) twihthe which at C), B locomotion A . P , shi 0.05; E ts1 – G D nerve )3D AB Journal of Cell Science a10codntsbl noyoi nsnpe 1025 synapses in endocytosis bulk coordinates Dap160 M opooyi eni oto av C n nNJ from NMJs in and (C) larva control in seen is an morphology (red) NMJ zones anti-Brp active outlines, presynaptic bouton reveal reveal to to (green) anti-Dlg extracts ( with actin. stained head and NMJ4 of Dap160 blot against western antibodies by with stained revealed as Dap160, produce neuronally B; and ( SH3A B. lacking UAS- and different SH3A expressing between flies region mutant linker L2 l SH3A; for used of loaded extract terminally the was of assay 1% pull-down anti-actin. the and for anti-dynamin with blotting western to P amounts. larger precipitate SH3A-D and and GST-SH3D GST-SH3C, whereas NV,Tkysps et cl as 2 bars: Scale ** test. s.e.m. post Tukey’s + ANOVA, mean indicate graphs Bar shown). infcnl ifrn rmta fcontrol, 25 of that from different significantly control, ( in larvae. boutons rescue satellite of numbers the between and different control the with in boutons ( satellite bouton. of satellite number one of example indicates arrow ( (D). i.4 rpriso the of Properties 4. Fig. Temperature-sensitive in detected was a10S3 n/rS3 rcptt yai from dynamin precipitate SH3B and/or Dap160-SH3A ˚ .( C. E H aelt otn r paeti the in apparent are boutons Satellite ) )At34 G D ˚ ,asgiiatrdcin( reduction significant a C, oooinbhvo in behavior Locomotion ) dap160 AB 0n (O). nm 50 ntemtn M pnsiuain ( stimulation. upon NMJ mutant the in ( rest. at NMJs mutant in immunolabeling dynamin of view the overall illustrating micrographs electron magnification the ( at (arrow). spots membrane in plasma Note proteins (J). both stimulation of after colocalization and (I) rest at olwn ihK zone high periactive following the pit at constricted accumulated labeled (arrow) a of image magnification yatcvsce z ciezn.Saebr:2 bars: Scale 0.5 zone. (A–J); active az, vesicle; synaptic Student’s mg hwn yai-rwti h Vpo in pool SV the dap160 within dynamin-ir showing image in (green) Eps15-ir (H). Eps15 unlike ( spots, in no accumulated is longer dynamin stimulation, similar During have (G). and distribution are rest (green) at Eps15 colocalized and largely (red) Dyn that showing ( NMJs. control in and rest Eps15 at (B) AP2 (Dyn), (C) dynamin (A) of localization ( en+sem * s.e.m. + mean r E p1-rad()A2i ncnrlboutons. control in AP2-ir (F) ( and Eps15-ir dynamin- (E) (D) ir, of redistribution a is there stimulation yatcvscepo rs)ada h A si)in (stim) PAZ and the at control and (rest) pool vesicle synaptic ( eiciezn nthe the in from zone dynamin periactive of Mislocalization 3. Fig. oprdt oto n fu and control to compared I A–C G P D dap160 , , J B , Q H ofcliae hwn P-r(e)and (red) AP2-ir showing images Confocal ) ulmtn.Teewr osgiiatdifferences significant no were There mutant. null akn HBadfl-eghrsu)pan- rescue) full-length and SH3B lacking , D uniiaino yai oaiaini the in localization dynamin of Quantification ) ofcliae of images Confocal ) ofclmcocp mgssoigthe showing images microscopy Confocal ) AB uata et ( rest. at mutant ulmtn osntmv t34 at move not does mutant null mutant. t ts.T -a;m iohnro;sv, mitochondrion; m, T-bar; T, -test. m dap160 KL;0.1 (K,L); m niDp6 bu,ist) Normal insets). (blue, anti-Dap160 d S ln ont oeta SH3AB that Note not. do alone GST P dap160 + , ( n Ipt.L,lne einN- region linker L1, (Input). 1 ane A eiiae rtiswr subjected were proteins recipitated stimulation. .5 *** 0.05; m dap160 uat a rpsindicate graphs Bar mutant. S-uinpoen containing proteins GST-fusion ) D dap160 dap160 m(C–E). , AB M dap160 4)i rdsursentered squares grid in 24%) rsu osrcs( constructs -rescue P N dap160 , High-magnification ) itreigo dynamin of Mistargeting ) n ullnt eceat rescue full-length and m llnt ecelarvae. rescue ll-length F .1 *** 0.01, dap160 M;0.2 (M); m C uatnreterminals nerve mutant uniiaino the of Quantification ) K ulmtn is mutant null compared lines rescue , P D D , ulmtn ( mutant null , AB L ofcliae of images Confocal ) uat( mutant w Low ) .0 using 0.001 1118 n full-length and D–F uatNMJs mutant P , edextracts, head B O .0 using 0.001 Following ) m D ˚ ) C(not (N); m High- ) AB D D dap160 D 1/Df). AB dap160 1/Df), mutant , m m Journal of Cell Science u espoone Fg E.T eti agtn fDap160 of targeting if test To 5E). (Fig. pronounced less Mistargeting but stimulated 5B,D,E). in (Fig. at NMJs dynamin control spots of to in contrast concentrated in is PAZ, longer the no dynamin is potassium, immunoreactivity dynamin of high 5A,C,E). images (Fig. with Confocal observed mislocalized. stimulation was upon Eps15 However, with a control, colocalization in As dynamin. strong of localization in differences significant niaigta Dap160 that indicating epnil o agtn n cuuaigdnmna ie of sites in at stimulation. recycling dynamin vesicle accumulating and synaptic predominantly targeting are for Dap160 responsible of indicate domains experiments dynamin-interacting these together, that Taken 5F,G). (Fig. control in h ao hsooia eet tro eprtr (22 temperature room at defects dependent. physiological temperature al., are major et defects The (Koh The 2004). construct al., et rescue Marie full-length 2004; the expressing in animals transmission synaptic in dap160 defects revealed experiments Earlier is in domains impaired NMJs dynamin-interacting in lacking uptake mutants dye Dap160 styryl and transmission Synaptic in perturbed is stimulation during PAZ the to development. during terminal nerve control the successfully to can transport module dynamin SH3A-B the the than portions other 1026 nml,w eoddi MCa mM 1 determine in transgenic after recorded in To we EJPs perturbed animals, Hz. is of 10 release amplitude neurotransmitter at in whether stimulation decrease high-frequency junction a miniature prolonged and of amplitude (mEJPs) average potentials in increase an included P n h Hdmi eino a10 togcolocalization Strong Dap160. of Dap160 against region of raised domain EH antibodies the and with AP2 NMJs stimulated double-stained nrsigsnpe fboth of synapses resting In ulmtnsadsoe htte a ersoe in restored be can they that showed and mutants null ora fCl cec 2 (4) 126 Science Cell of Journal D AB n P nsoso loecnewsobserved was fluorescence of spots in AP2 and D AB D eoaeaogwt P,a observed as AP2, with along relocate B D uatsnpe a loevident, also was synapses mutant Drosophila B and 2+ D AB n bevdn significant no observed and D AB aveceryso that show clearly larvae ave hr eeno were there larvae, Msuo intense upon NMJs D AB uat,we mutants, D D ˚ AB AB C) shi Fg AC.Teapiueo vkdEP eodda . Hz 0.3 6D,E). (Fig. at altered recorded significantly EJPs not evoked also of was mEJPs amplitude spontaneous The of 6A–C). frequency (Fig. and amplitude the in difference tmltdfr4mntsa 0H n hnsnl stimuli single amplitudes EJPs then recovery. and the Hz measure 10 to Hz in at 0.3 minutes at 4 al., delivered et for (Koh stimulated mutants been null in has studies stimulation previous 2007). our high-frequency average when in in during decrease observed stimulation similar amplitude A during 6F). (Fig. EJP amplitude control the EJP to average compared in decrease vnsdsperd6 eod fe h tmlto Fg 6I). (Fig. stimulation fusion large the These after the 6G,H). control seconds (Fig. 60 during mV large than 11.2 disappeared of to observed events some rate Interestingly, up stimulation, reached were slower high-frequency which S5A). following much events phase recovery a Fig. spontaneous at amplitude material and (supplementary minutes 5 than easse odn ftesyy y M-3i control, in FM1-43 dye styryl the of after loading assessed briefly we and during synapse the activity. in following high-frequency Slow machinery events and/or release endocytosis transmission. spontaneous in are the defect large a synaptic domains suggest of stimulation binding high-frequency normal presence dynamin and maintain that recovery to indicate data necessary the Hence, inda 0scn nevl n omlzdt h is EJP were first data the to recorded The normalized The and bin. minutes. were intervals 10 amplitude 60-second 6/7 for at Hz Muscles binned 10 at release. stimulated neurotransmitter high-frequency ttsial infcn eraei M-3laigi the in a loading observed FM1-43 We in 2008). decrease al., D significant et (Verstreken statistically protocol standard AB ete eemndwhether determined then We osuytercvr rmsnpi ersin Mswere NMJs depression, synaptic from recovery the study To ofrhrasyteosre eeti yatctransmission synaptic in defect observed the assay further To dap160 ts1 n full-length and nml scmae ocnrladfl-eghrescue full-length and control to compared as animals D AB ecemtnsrcvrt h rgnllvli less in level original the to recover mutants rescue t en+sem * indicate s.e.m. graphs + Bar mean spots. to concentrated and and control both In anti- fcnrl()and (F) control of ofiin PC.( correlation (PCC). mutant. Pearson’s coefficient rescue as the measured in is lower expression Colocalization slightly 160 to Dap due of possibly level is control rescue to stimulation. full-length compared upon in and level rest colocalization at Lower lines, rescue different and p1-ri otn rmcontrol, from boutons in Eps15-ir o ocnrtdt pt n sdsesdi h bouton. the in dispersed is ( and spots to concentrated not the expressing larvae the of D of terminals perimeter Nerve the (C,D) to bouton. close spots stimulation, to following concentrated and, colocalized Eps15-ir largely and dynamin-ir are larvae, control and In (Dyn) (A,B) dynamin Eps15. against antibodies with labeled emnl ie trs n pnhg K high upon and rest at fixed terminals o orc yai agtn uigsynaptic during required activity. are targeting Dap160 dynamin of correct domains B for and SH3A 5. Fig. ts.Saebr 2 bar: Scale -test. E Brsu osrc.Atrsiuain yai-ris dynamin-ir stimulation, After construct. AB-rescue uniiaino ooaiaino yai-rand dynamin-ir of colocalization of Quantification ) a dap160 dap160 aatn(P)adat-a10atrstimulation. after anti-Dap160 and (AP2) -adaptin ( A – D D ofclmcocp mgso nerve of images microscopy Confocal ) ecetidisa aveuiga using larvae instar third rescue AB P , D dap160 m yassso significant a show synapses AB .5 *** 0.05; D F m. , AB G G ev emnl aee with labeled terminals nerve (G) ofclmcocp images microscopy Confocal ) aeigi otycolocalized mostly is labeling , D P AB , .0 sn Student’s using 0.001 Mscnsustain can NMJs dap160 + stimulation ul( null D D 1/Df) AB , Journal of Cell Science aeilFg 6)adtettlnme fvsce e terminal high per supplementary vesicles to of 7A,B; number (Fig. exposure total rescue the minutes and and of S6A) 10 control Fig. morphology material after both the to and rest, similar rest At at potassium. fixed instar were Third control, stimulation. from during formed origin larvae structures the internal investigate the to microscopy of electron domains performed dynamin-binding next lacking We mutant Dap160 structures the membrane in internal of organization Subcelluar Ca mM 1 to returned the were when These disappeared larvae terminal. was compartments the dye membrane in the labeled compartments FM1-43 of membrane some large that in supplementary suggesting trapped 6N–P; S5B–D,H,I), (Fig. Fig. internal of observed material number was the in structures increase fluorescent an addition, In 6J–M,Q). (Fig. r eae otesnpi eil yl splmnaymaterial (supplementary cycle vesicle S5E–G). synaptic Fig. the to related are tmltdwt ihK high with stimulated D AB + uprigtepooiinta they that proposition the supporting , , D B n ullnt eceanimals rescue full-length and , 2+ D ouinadagain and solution AB Msi very is NMJs a10codntsbl noyoi nsnpe 1027 synapses in endocytosis bulk coordinates Dap160 ek otelremmrn naiain eeotnseen often in present were also membrane invaginations were invaginations stimulated membrane narrow membrane Bulk large PAZ by 7E–H). (Fig. the the connected In in to the vesicles observed sections. necks to Large were ultrathin attached 7D–J). connections (Fig. invaginations serial tubular membrane by by membrane bulk verified large large Numerous as 7C,D,L). addition, (Fig. appear, reduced strongly vesicles is pools distal vesicles, observed D docked were of structures number endosome-like the 7J,M). and 7K). (Fig. (Fig. in pits unchanged increase remained endocytic while T-bar reduced, significant the was pool to No distal proximal the pool in SVs the of density packing the eghrsu n oto.Cae isfudi yassfrom synapses in found pits Coated control. and among evident rescue was length pits collared or pits endocytic ( different significantly not is AB olwn tmlto,daai tutrlcagsocrin occur changes structural dramatic stimulation, Following upiigy osgiiatdfeec ntenme fcoated of number the in difference significant no Surprisingly, Ms h est n ubro V npoia and proximal in SVs of number and density The NMJs. D B yass(upeetr aeilFg S6B). Fig. material (supplementary synapses eoddfrom recorded hn5m uig1mnt olwn high-frequency following minute ( 1 stimulation. during mV 5 Hz ( than 10 minutes. following 10 minute for 1 stimulation during mEJPs of from amplitude recorded ( mEJPs normal. stimulation. are Hz control 10 of minutes 10 tmlto o 0mnts omlzdt h is EJP first the to ( normalized amplitude. minutes, 10 for high-frequency stimulation at amplitude Evoked depression. Hz. 0.3 at recorded ( EJPs ( amplitude stimulations. average Hz showing 0.3 by induced EJPs et(,) rAOA ue’ otts Q.* (Q). test post Tukey’s ** ANOVA, or (F,I); test s.e.m. + mean Student’s indicate using graphs Bar intensity. labeling mltd rfeuny ( frequency. or amplitude and Control mEJPs. of D frequency and amplitude average aesbuoso h M ncnrladfull-length and in control labeling in whereas NMJ rescue, the of boutons labels aeigi high-K in labeling D uln h otnbres ( lines borders. Dashed bouton perimeter. the is bouton outline dye the in larvae to whereas (P) close O), rescue observed in full-length (arrow and bouton (N) the control of lumen in the uptake in dye that Note weaker. oto and control stimulation. ( as upon well uptake as membrane phase, decreased recovery during by mEJPs followed large stimulation high-frequency at depression in Synapses 6. Fig. F A AB AB hnsiuae t1 Hz, 10 at stimulated When ) P xml rcsfrmJsrcre rmmsl of 6 muscle from recorded mEJPs for traces Example ) , ontso infcn ifrnei EJP in difference significant a show not do n ullnt ecelra fe 0mntsof minutes 10 after larvae rescue full-length and .1 *** 0.01, G D J–P AB rcssoigeape flremEJPs large of examples showing Traces ) D P P t Bdrn h is 5scnsfollowing seconds 15 first the during AB . , ts BCEH;AOA Bonferroni ANOVA, (B,C,E,H); -test M-3deutk ycontrol, by uptake dye FM1-43 ) M.( NMJ. + .0.Saebr:1 bars: Scale 0.001. .5 Student’s 0.05; ouina 34 at solution H D AB itga hwn h average the showing Histogram ) B D , xml rcso h evoked the of traces Example ) uatso synaptic show mutant C shi Q itgassoigthe showing Histograms ) I ubro EP larger mEJPs of Number ) uniiaino FM1-43 of Quantification ) ts1 D AB D and ˚ AB .F14 brightly FM1-43 C. Msso synaptic show NMJs D t ts) However, -test). Msaccumulates NMJs AB E m D a graph Bar ) m. AB ssignificantly is , D P B , full- , 0.05, shi ts1 , Journal of Cell Science Ms(i.7) nsmay Maayi eeldta deletion in and that domains revealed control binding analysis dynamin EM stimulated of summary, In vesicles in 7M). docked different (Fig. NMJs these significantly of not number was The active as intermediates. cryosubstituted assemblies endocytic the membrane, vesicle of plasma in studies docked zones the tomographic represent by in they described formed that not suggesting are dense electron these projections the that showed Tilting dense microscope electron S6C,D). the dense in Fig. electron protrusions mutant material and by control zone (supplementary in active both NMJs synapses the (Koh in to observed reported were attached been projections SVs has 2004). stimulation al., upon et pits coated of number different is This S6E). Fig. from material (supplementary shape in vary D 1028 AB , dap160 D B eceadcnrlalhdauiomsz n i not did and size uniform a had all control and rescue , Drosophila ora fCl cec 2 (4) 126 Science Cell of Journal ulmtn Msi hc oeaeices in increase moderate a which in NMJs mutant null Ja ta. 00)adaeteeoenot therefore are and 2010a) al., et (Jiao dap160 ed osvr eet in defects severe to leads D AB ukmmrn paea h A uighg-aesynaptic high-rate during PAZ the the that at and activity uptake membrane bulk itaeiua arx ihntenretria opeetits or prevent cytoskeletal to terminal unknown nerve an the with that within noted associated matrix’ have ‘intravesicular is studies dynamin Other suggesting activity (Marie 2006). that synaptic rest al., during at changes et synapses localization in Wagh dynamin PAZ 2004; the membrane for al., be marker dynamin et a for can clathrin- as labeling used of dynamin Henceforth, been 1995). has necks that al., the et confirming (Takei to associated thus and membrane pits, plasma coated the distinct to at dynamin PAZ. regions the localized at have recycling studies membrane implicated SV Immunocytochemical enzyme during constriction reaction fission key the the in is dynamin GTPase The Discussion membrane. presynaptic the from vesicles clathrin-coated EGH;2 m() 5 m(I). nm 250 (F); nm 25 (E,G,H); sn Student’s using rpsidct en+sem ** s.e.m. + Bar mean . indicate ax, graphs profile; endosome-like el, vesicle; control in different significantly and not were pits endocytic tmlto ( stimulation B ev emnl hwta trs h est of density the rest at that show terminals nerve (B) ( of in terminals and structures nerve vesicles endosome-like synaptic large of of number accumulation in Decrease 7. Fig. ev emnl qatfe nK.( of K). K pool in distal (quantified the terminals in nerve decreased is vesicles synaptic h rxmladdsa oea et( rest at zone distal and proximal the ( stimulation. ( profiles H). ( and (arrow). F structure in (arrows and membrane E plasma ( in the (arrowheads to other and each G) to connected profiles often endosome-like are that show images ( magnification D profile). in endosome-like (arrow one observed indicates also are profiles Endosome-like distal and in proximal both zones in decreased is vesicles synaptic A I mg hwn xml flreendosome-like large of example showing Image ) + , B tmlto teeae eprtr,tenme of number the temperature, elevated at stimulation D lcrnmcorpso oto A and (A) control of micrographs Electron ) D AB AB ,Tbr ,mtcodin v synaptic sv, mitochondrion; m, T-bar; T, . . D AB 5 m sicesdin increased is nm) 150 uatde o nii h iso of fission the inhibit not does mutant L K,L .( ). ev emnl qatfe nL). in (quantified terminals nerve t ts.Saebr:0.5 bars: Scale -test. M uniiaino yatcvsce in vesicles synaptic of Quantification ) J h ubro okdvsce and vesicles docked of number The ) h ubro ag endosome-like large of number The ) dap160 D AB P ece larvae. rescued , C D .1 *** 0.01; , K AB m D E–H AD;0.1 (A–D); m n following and ) olwn high Following ) following Higher ) P , D AB 0.001 D AB m m Journal of Cell Science lse trs n trlctst h Asdrn synaptic during PAZs is the vesicle domains the to SH3A-B in relocates via localized it interaction is upon and Dap160–dynamin dynamin changes activity. rest of synapse at pool we the cluster large Furthermore, in A event. we localization this activity. techniques dynamin 1996; of that high-resolution al., origin show Using et subcellular (Estes 2010). the sites resolved al., presynaptic et the Pechstein from away diffusion uat yai sn ogrtree otepratv oe nta tis and it invaginations accumulated. instead membrane are zone, Large structures periactive terminal. endosome-like the nerve to the targeted in longer dispersed no is In dynamin (ADBE). clathrin-mediated mutant, endocytosis by bulk synaptic activity-dependent recycled and intense is (CME) during membrane endocytosis zone vesicle periactive synaptic from the Fused redistributed to activity. are cluster dynamin vesicle and synaptic Dap160 the Eps15, periactive AP2, the cycle. proteins to vesicle endocytic targeting synaptic dynamin the for mechanism during the zone of Scheme 8. Fig. in at activity dynamin synaptic of during PAZ accumulation the and re-localization the for important rmtegnrlrdcino yai eesin levels that dynamin show studies of Our reduction mutants. general the from in dynamin moreover of and mislocalization with phenotypes, the developmental experiments the from Our dissociated be development. during dynamin NMJ D of regulation the the synaptic at in 2004), al., involved levels also al., et of is et it (O’Connor-Giles addition, Marie defect In maintenance 2004; 2008). developmental al., a et the represent (Koh which boutons in satellite small role in important In architecture. an plays idn odnmn copihtefntoslne othe in levels to dynamin linked affect ultimately direct functions and its NMJ the to with the accomplish addition of Dap160 in maturation dynamin, recycling, of to SV interactions in binding that involved propose not as dynamin proteins We of level NMJs. same the control maintain and morphology synaptic AB rvossuishv lal eosrtdta a10also Dap160 that demonstrated clearly have studies Previous uat hwta h ucinledctcpeoye can phenotypes endocytic functional the that show mutants dap160 uat,teNJdsly nincrease an displays NMJ the mutants, D D AB nwl-ye(t nml the animals (wt) wild-type In Drosophila AB otn ipa normal a display boutons yasscnb separated be can synapses yass(i.8). (Fig. synapses dap160 D null a10codntsbl noyoi nsnpe 1029 synapses in endocytosis bulk coordinates Dap160 AB P,adA3a etaddrn tmlto ean obe to remains stimulation during and clathrin, of rest Our localization the at The to PAZ. investigated. AP3 mutant. recruitment the the and Dap160 in at AP1, and affected recruitment not Eps15 is clathrin AP2, PAZ the that show to studies very prior at classical step inhibited the becomes early that pathway is Cousin, endocytic bulk and possibility (Cheung non-exclusive clathrin-mediated different of AP3 other utilizes and The step AP1 it 2012). as contrary pathway a such but complexes, clathrin-mediated clathrin, at adaptor involve classical Recent may occurs the SVs. step of to this recycling formation that the structures suggest SV to studies is leads endocytic of that possibility pathway One block occur? this the the does that How from are NMJs. SVs in reformed that accumulated of suggest properly endosomes Accumulation nerve not and in stimulation. invaginations vesicles high-frequency synaptic membrane of upon number the terminals in with 2004; reduction 2008). interacts al., dramatic al., et wreck et (Rodal domain Nervous (Coyle SH3A its 2008). module via al., dynamin is domain et with partner SH3C-D interact O’Connor-Giles to binding shown Dap160 been such has the which One wreck, Nervous development. probably during synapses oan.Itrsigy thsbe oe ale that earlier noted SH3 been large in dynamin-binding has structures dramatically of membrane lacking bulk it number of mutants were appearance Interestingly, the the structures and domains. in ‘necks’ endosomal increased presynaptic thin the interconnected to with linked invaginations membrane number The membrane perturbed. uncoated is of intermediates clathrin- endocytic classical bulk the of the of fission to step Dap160 fission in the pathway by for mediated This dynamin essential of inhibited. not targeting is not PAZ when the is that increase pits implies dynamin- clathrin-coated significant further that of a supports budding latter observe The mediated synapses. not control did to compared we and mutants agtn fdnmnaepriual motn o synaptic for proper important and activity. synaptic particularly high-rate Dap160/intersectin are during function of dynamin of function targeting ‘traffic scaffolding represent synapses the scaffolding the at the in trafficking possibly imbalanced because membrane were bulk occurred experiments controlling dramatic have events components may to fusion our which Giant lead accidents’, synapses. in will in release vesicles observed quantal large of of membrane distortions clathrin-mediated by Fusion fused probably of most the endocytosis. cessation SVs into that after reformed implies vesicles becomes active large the stimulation at these high-frequency fuse of stimulation Disappearance upon vesicles zone. terminals bulk nerve that in suggesting observed activity, the high-rate in following phase supports endocytosis bulk this the and All coordinate clathrin-mediated clathrin synapses. 2008). with between 1 together al., balance bulk dynamin dynamin mouse massive et that in hypothesis A reported (Hayashi 2008). been also knockouts al., has controlling et uptake in or (Kasprowicz or membrane chain function heavy mutants dynamin clathrin of clathrin to inactivation upon linked function dynamin mutants in occurs h eraei ertasiso in in decrease The ltrncae isd o emafce in affected seem not do pits Clathrin-coated ag pnaeu Jswr eodddrn h recovery the during recorded were EJPs spontaneous Large Drosophila D AB uat hsfrhridctsthat indicates further This mutant. yass oee,teproper the However, synapses. D AB slkl u othe to due likely is Drosophila D AB rescue NMJs Journal of Cell Science uat n rngnsadrfre oi h eta ‘control’. as text the in to referred and transgenes and mutants sRgo fItrs.Maueet ae ncnoa mgswr rm15 genotype. from and were condition each images was for for confocal animals selected used coefficient on different were three based that least was correlation Measurements at boutons from Interest. individual Volocity Pearson’s boutons of of were (PerkinElmer). sections Region colocalization. optical measurements as Volocity middle of for All or determined levels (Adobe). ImageJ evaluating and ImageJ in Zeiss), Photoshop using (Carl adjusted 700 performed were LSM or 63 contrast a with and or captured (http://rsb.info.nih.gov/ij) 510 Brightness were LSM software. images using accompanying confocal objectives the Other immersion to oil nm. calibrated NA 70 was 1.4 to which a up Microsystems), with resolution (Leica equipped provide Software system Suite 63 microscope Application using confocal Leica Microsystems) SP5 (Leica on unit STED collected were S1B,C Fig. medium mounting Vectashield in mounted at used Laboratories). were was (Vector Samples previously antibody secondary (Leica). were or conjugated described Cy5 1:100 R1 555 to dye dilution as 488, conjugated Otto antibodies (Invitrogen). Alexa performed 1:100 secondary to at 1:500, used was conjugated at used antibodies were fillets Secondary (Invitrogen) 647 2008). larval al., instar et (Verstreken third image of fluorescent of Labeling quantification and imaging Immunohistochemistry, The pig). (guinea in 1:1000 NMJs and and (rabbit) tissues 1:500 label dilutions: not in did used (Koh in antisera were characterized 2007) previously antisera al., Dap160 et pig guinea and rabbit Polyclonal Antibodies For Healthcare). (GE sepharose 25 glutathione pull-down using GST protocols purified and standard cells to BL21(DE3) in according expressed were proteins PRD GST-fusion dynamin GST-fusion or GST The pull-down GST of w generation and stocks Fly Methods and Materials 1030 dap160 niBpadA2atbde eekn it rmE uhe Uiest of (University Buchner E. from gifts kind anti-Eps15, were pig 1993). guinea antibodies al., 2006); Wu et 1994); AP2 (Littleton al., al., 1:1000 and et anti-synaptotagmin et rabbit (Wagh Anti-Brp 2007); (Zinsmaier 1:100 al., 1:50 et anti-Brp, (Koh 49, mouse 1:1000 mAb 2000); anti-CSP al., et mouse (Cho 1:2000 anti-Dlg, anti- rabbit Ja dilutions: and following the in used or . l h xrcswr nuae nartto he o or t4 at hours 4 for wheel of rotation volume a total on a incubated in were (Sigma) extracts Cocktail The Inhibitor ml. Protein 0.7 with supplemented 100) aallwt oto and control with parallel MgCl mM 2 KCl, mM 100 HEPES, mM of (20 mg buffer 2 to added Healthcare), (GE beads nsplmnaymtra al 1 h C eunei dnia ooewas one to identical is (Fusion sequence product ACD pUAST- The the PCR sequences S1. in ended primer deleted Table all blunt material See supplementary plasmid. of ABCD in self-ligation from amplified by ACD Finnzyme), The polymerase, generated S2). Table was material (supplementary construct IN) Bloomington, Center, Resource MreI– or PshAI SpeI– pUAST- or pUAST- the into SpeI–PshAI- of inserted The sites S1). were PshAI Table fragments material PCR (supplementary letters MreI–PshAI-digested bold in shown are primers forward PCR- D was with FBgn0023388) ID FlyBase amplified 3586–4221, (nt SH3B downstream sequence GFP AEgl(nirgn n muobotduigseii antibodies. specific 2 using SDS- with immuno-blotted 4–12% a and eluted on (Invitrogen) them were gel resolving complexes by PAGE analyzed Protein and (Invitrogen) buffer. buffer PD sample PAGE with times four washed a bandfo h loigo rspiaSokCenter. Stock Drosophila Bloomington the from obtained was epciey(lBs DFc0046adFBcl0169663, and FBcl0001466 ID and using IP14822 (FlyBase PCR clone respectively by cDNA obtained Dap160 template were a Healthcare) as (GE vector pGEX-6P-2 in dynamin-PRD respectively. of upstream nt 156 or nt 579 located extensions nt 18 for or region nt amplified 48 had fragments Df(2L)bur-K1 04.Frrsu xeiet h a-erldie line driver pan-neural the K1 experiments rescue For 2004). 1118 HAruigtepUAST- the using SH3ABr TDadcnoa irsoyiae o i.1 n upeetr material supplementary and 1D Fig. for images microscopy confocal and STED ognrt h A-ececntut ihS3 n HB(pUAST- SH3B and SH3A with constructs UAS-rescue the generate To h H xrsincntut 1AL,L-,A,AC,C ,and D, C, ABCD, AB, L2-B, L1-A-L2, constructs expression SH3 The rbr,Gray n .Gonza M. and Germany) ¨rzburg, / w CyO ; ; cl,19) os nidnmn :0 coe4;B isine) rabbit Biosciences); BD 41; (clone 1:300 anti-dynamin, mouse 1997); ¨ckle, a sda a10wl-yecnrlwt h aegntcbcgon as background genetic same the with control wild-type Dap160 as used was UAS-dap160 dap160 D , dap160 AB twi-GFP rwt HB(pUAST- SH3B with or ) ora fCl cec 2 (4) 126 Science Cell of Journal D srfre oas to referred is 1 D /CyO B ae59n r16n eein opiigS3Bo SH3B, or SH3AB comprising deletions nt 156 or nt 579 have , WT eecosdto crossed were m fcrepnigpoenwr on oguahoeagarose glutathione to bound were protein corresponding of g D , n 1nnGPlra eeslce o experiments. for selected were larvae non-GFP F1 and Band AB dap160 twi-GFP shibire dap160 dap160 dap160 D ts1 dap160 D ; .Alcntut eevrfe ysequencing. by verified were constructs All B. dap160 ,rsetvl.Rsrcinstsfrsub-cloning for sites Restriction respectively. B, UAS-dap160 dt o hw) ae fohratbde was antibodies other of panel A shown). not (data S3L--2L-///BADAC)and -SH3(L1-A-L2/L2-B/C/D/AB/ACD/ABCD) eutn lsis pUAST- plasmids, Resulting . ´ D UAS-dap160 lez-Gaita HAfor SH3ABf DApamda epae eutn PCR Resulting template. as plasmid cDNA uli ecie nKhe l Khe al., et (Koh al. et Koh in described is null Drosophila rngn n xrsinconstructs expression and transgene dap160 6 a ´ dap160 i meso betv 14N)and NA) (1.4 objective immersion oil aatn P,15 (Gonza 1:50 AP2, -adaptin, Gnv nvriy Switzerland). University, (Geneva n D AB2 shibire D D B1 B 2 8B D eee oan,genomic domains, deleted ) MPS,05 rtnX- Triton 0.5% PMSF, mM 1 , 5C edetati uldw (PD) down pull in extract head ulmtnswe tie in stained when mutants null HB n ees primer reverse and SH3Bf ; or DAcoeL262shi, LD21622 clone cDNA dap160 w C155 ; Drosophila dap160 - D Gal4 1 shibire dap160 /CyO D ; 1 ´ Df(2L)bur- /CyO , dap160 lez-Gaita Genomics ts1 twi-GFP D 6 ˚ AB ,then C, ( shi , SDS- twi- and ts1 D 6 ´ 1 n ) / , , cieznsfo he nml eeue o quantifications. for 10 particle least used gold At the were S2A). to animals Fig. PAZ three material the (supplementary from in PAZ PAZ in zones to density shown PAZ/outside active was adjacent particle area as ratio gold PAZ a the of membrane density and in PAZ. ratio density plasma zone particle the and the active as Gold SVC from calculated the S2A. was the lumen to Fig. the adjacent in synaptic material into nm densities supplementary 500 space or particle PAZ area cytosolic the gold PAZ membrane nm in 100 of plasma density at the particle sum gold as (%) the of defined ratio to particles the SVC as gold calculated or was of (SVC) pool distribution vesicle Relative (Microsoft). Ca without HL3 in prepared were larvae instar third two from from NMJs Fillets TEM 6–9 and immunocytochemistry genotype Preembedding an each at For Confocal A2–A5, nm. scored. segments 0.5 NMJ4, and of from boutons ( thickness above from satellite or described section axis as as NMJ optical classified major obtained the were were from anti-Brp. images branch bouton with al. that et labeling terminal anti-Dlg. boutons Marie Small by to with the 2004). according detected labeled al., performed were et was were number (Marie zone fillets bouton active satellite larval of the instar Quantification of third T-bars outline, Presynaptic bouton reveal To morphology Bouton uiga3mnt eidwsmaue ypaigtePtids vragi of grid a over dish Petri cm the (8.5 placing dishes Briefly, by larva Petri each ). measured of by was al. travelled centre period Distance et agarose. the 3-minute (Yang 1% in a with al. coated during placed height) et were cm Yang 1.5 larvae diameter, to instar according third tested individual was locomotion Larval assay locomotion Larval and KCl mM 60 with HL3 in 34 were incubated a larva in and test one plates and Sylgard control 4 one al. small experiment et on each Verstreken For by dissected described 2008). as al., performed et were (Verstreken experiments uptake dye FM1-43 uptake dye FM1-43 Ca in dissected were larvae instar Third Electrophysiology 2010b). 2k al., et a mounted (Jiao using described bottom traced 8.0 earlier were Maya as using contours into rendered Membrane transferred photographed (TVIPS). and Camera digitizer were CCD F224 sections TemCam ultrathin images Serial TEM of reconstruction 3D Ca y nodn w eaaeeprmnsfrec eoyewr are out. carried were genotype test al., To each et data experiments. for uptake as experiments (Verstreken dye well separate in described two as tested unloading were acquisition as genotype dye Data performed per Zeiss). animals were (Carl Three 2008). 700 quantification LSM and a processing on 1.0) (NA objective rcse sn rp a rs 5 eodnswr rm51 nml per animals 5–11 from data were and Recordings software v5. v10 condition. Prism Clampfit the and for Pad using stimulation genotype measuring Hz Graph analyzed 10 by were using minutes, recordings 4 processed analyzed following All Hz was traces. or 0.3 five at five stimulation for evoked stimulation EJPs the from Hz six calculating of 10 by amplitude Recovery of analyzed bin minute were recordings. every EJPs 1 per more Evoked EJPs during mEJPs traces. 10 value period. of five average amplitude recording of the membrane average the minimum taking a by during the amplitude for the mV and when when min 10 frequency EJPs than for only evoked less for quantified and analysis were by measurement fell for mEJP in potential used for mV used membrane was 60 Instruments) Low were above ( was muscles. interface Recordings potential A/D on were the running collection. an Data software with Hz. v10 data innervating equipped 10 ClampFit computer at and PC amplifier, frequency 2B a high motor Axoclamp Hz, an 0.3 stimulate using at recorded applied to was stimulation used frequency was solution ihK high 3wt hnwle irppte ildwt C nH3slto containing solution HL3 segment in in KCl 7 M CaCl or 3 mM 6 with muscle 1 filled from micropipettes recorded thin-walled were with mEJPs A3 2007). al., et (Koh previously 0mnts etn odtos rsiuae yadto f6 MK mM 60 of addition for by EDTA stimulated either or containing buffer conditions, HL3 resting in minutes, incubated 10 were fillets experiments, ahdwt L ouincnann MCa mM instead 1 was containing fillets solution larval experiments HL3 unloading with For washed minutes. 15 over solution HL3 mgJsfwr,adsaitcleauto a efre sn Excel using performed was NIH evaluation knife using diamond quantified statistical were a Images and with (FEI). examined cut and microscope software, grids, electron were on 12 citrate ImageJ sections described lead Tecnai and earlier ultrathin a acetate as uranyl with Serial EM 1% for with 2010b). stained embedded (Diatome), M al., and 0.1 labeled et in and solution, (Jiao paraformaldehyde 7.2 4% pH in buffer, fixed were phosphate specimens The minutes. 10 dap160 m 2+- M-3(nirgn akt aki . lEpnoftb o 0minutes 10 for tube Eppendorf ml 1.5 a in back to back (Invitrogen) FM1-43 M reH3slto.Lbln a atrdwt 40 a with captured was Labeling solution. HL3 free + ˚ aebt.Ecs y a hnwse wyrpael ihCa with repeatedly away washed then was dye Excess waterbath. C xetti iewtotF14 y,adte ujce owsigwith washing to subjected then and dye, FM1-43 without time this except ul rtre(l te eoye)lra eeanalyzed. were larvae genotypes) other (all three or null) 2 Hlemn ta. 00.Ascineetoebcfle ihHL3 with backfilled electrode suction A 2010). al., et (Hallermann H Drcntuto rga n surface and program 3D-reconstruction 2+ reH3slto sdescribed as solution HL3 free 2+ n hnaansiuae with stimulated again then and 6 ae immersion water 2+ 2 + For . + -free 6 for TM 2k Journal of Cell Science egsn .M n eCmli P. Camilli, De and M. S. Ferguson, vrrn . a,H,Wlhr . udogr . oii,N n Shupliakov, and N. Tomilin, A., Sundborger, K., Walther, H., Gad, E., Evergren, se,P . os . a e le,A,Kly .B,Kiha,K .and S. K. Krishnan, B., R. Kelly, A., Bliek, der van J., Roos, S., P. Estes, oaHbioaBn o antibodies. for Bank Hybridoma Iowa 60(rpSa otae a ig,C) o-infcn ifrne r not are differences or Non-significant v5.0 CA). Prism selected figures. Diego, GraphPad in comparing San using indicated test, performed Software, variance with was comparison (GraphStat of analysis mean multiple v6.0 Statistical every analysis Bonferroni means. comparing one-way of or post-test, pairs groups mean, Tukey’s with other two either every than used, more was (ANOVA) between differences the ita,J n yn .A. T. Ryan, and J. Dittman, oooinwstse t25 at tested was locomotion ttsia nlsso w ruswseautduigStudent’s using evaluated was groups two of analysis Statistical Statistics mm 25 ol,I . o,Y . e,W . ln,J,Fretd . iteo,J .and T. J. Littleton, T., Fergestad, J., Slind, C., W. Lee, H., Y. Koh, P., I. Coyle, W. K. Choi, and S. Izaddoost, J., Chern, O., K. Cho, A. M. Cousin, and G. Cheung, K. Broadie, References Medical at http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.118968/-/DC1 online Hughes available Howard material Supplementary the Hja of investigator and an Foundation Institute. is HEALTH-F2-2009- Wallenberg H.J.B. number the O.S.). and agreement 242167]; [grant Programme Framework [grant Seventh (SynSys-project) Union Council European Research the Linne DBRM; Swedish and Center 529-2009-6646/ESF-Euromembrane] the and by 13473 supported numbers was work This Funding experiments; performed experiments; A contributions Author Gonza M. Buchner, E. thank We Acknowledgements ltsi uiiidcabr eoelcmto a etd o ahgenotype each For tested. tested. were agar was larvae juice locomotion 24–27 apple before condition yeast-supplemented chambers and preheated humidified on in minutes plates 10 for preheated were ..aaye aa l uhr icse h aaadcmetdon commented and data the manuscript; discussed the authors all data; analyzed O.S. ˚ MEW,OV,KAR,TWK,HJB n ..designed O.S. and H.J.B. T.W.K., K.A.R., O.V., .M.E.W., GTPase. noyi oei h eta synapse. central the in zone endocytic O. otn eaiet oprmn-pcfcmarkers. compartment-specific to relative boutons M. Ramaswami, terminals. euae yatcgot nDrosophila. in growth synaptic regulates B. Ganetzky, Drosophila. in patterning disc eye 342. for essential is membrane peripodial Biol. o eeaino yatcvsce rmatvt-eedn ukendosomes. bulk activity-dependent from vesicles synaptic Neurosci. of J. generation for 20) nesci sangtv euao fdnmnrcutett h synaptic the to recruitment dynamin of regulator negative a is Intersectin (2007). 2 14 qae n crn h ubro rdsursetrd Larval entered. squares grid of number the scoring and squares R853-R855. , a.Rv o.Cl Biol. Cell Mol. Rev. Nat. 20) yas cfodn:itreto fedctssadgrowth. and endocytosis of intersection scaffolding: Synapse (2004). nu e.Cl e.Biol. Dev. Cell Rev. Annu. 32 20) evu rc,a H dpo rti htitrcswt Wsp, with interacts that protein adaptor SH3 an wreck, Nervous (2004). A ˚ 6014-6023. , MEW,WJ,OV,KAR,ES,KLS n O.S. and K.L.S. E.S., K.A.R., O.V., W.J., .M.E.W., A 19) rfi fdnmnwti niiulDoohl synaptic Drosophila individual within dynamin of Traffic (1996). ˚ MEW n ..woetemanuscript. the wrote O.S. and .M.E.W. 21) dpo rti opee n r essential are 3 and 1 complexes protein Adaptor (2012). ˚ A n 34 and C ˚ 20) oeua icir fedctssa nerve at endocytosis of circuitry Molecular (2009). MEW,WJ,OV,KAR,ES and E.S. K.A.R., O.V., W.J., .M.E.W., 13 75-88. , 25 .Neurosci. J. 21) yai,amembrane-remodelling a Dynamin, (2012). ´ 133-160. , ˚ Neuron lez-Gaita .A lvtdtmeauetelarvae the temperature elevated At C. 41 .Neurosci. J. ´ 27 521-534. , 20) oe inln rmthe from signaling Novel (2000). n h nvriyof University the and n 379-390. , 16 t tss oevaluate To -tests. rfne (to ¨rnfonden 5443-5456. , Cell 103 331- , Curr. a10codntsbl noyoi nsnpe 1031 synapses in endocytosis bulk coordinates Dap160 ´ apoiz . unn . ikeiz . aes .L,Siz .and L. Smitz, L., R. Habets, K., Miskiewicz, S., Kuenen, J., Kasprowicz, io . hpikv .adSulao,O. Shupliakov, and A. Shupliakov, W., Jiao, Franze S., Masich, W., Jiao, en,W . oco,E,Miek,M,Eege,E,Vli,Y,Mta,R and R. Mittal, Y., Vallis, E., Evergren, M., Meinecke, E., Boucrot, M., W. Henne, Gonza isae,K . bre .K,Bcnr . atr .adBne,S. Benzer, and N. Walter, E., Buchner, K., K. B. Eberle, M. E., Sokolowski, K. and Zinsmaier, J. A. Hilliker, A., S. Shaver, P., Yang, aah,M,Riod,A,OToe . aaie . ols,C,Ceoa O., Cremona, C., Collesi, S., Paradise, E., O’Toole, A., Raimondi, M., J. Hayashi, R. Kittel, and M. Heckmann, S., Hallermann, ah .A,Rse .M,Aa,E,Hfae,A,Shekr,I,Du I., Schwenkert, A., Hofbauer, E., Asan, M., T. Rasse, A., D. Wagh, J. H. Bellen, and T. Ohyama, P., Verstreken, ae,K,MPesn .S,Shi,S .adD ail,P. Camilli, De and L. S. Schmid, S., P. McPherson, K., Takei, P. Camilli, De and Y. Saheki, B. R. Kelly, and J. Roos, B. R. Kelly, and J. Roos, ong .H n kd,K. Ikeda, and H. J. Koenig, oa,A . ooaBre,R .adLtltn .T. J. Littleton, and N. R. Motola-Barnes, A., A. Rodal, iteo,J . eln .J n ei,M S. M. Perin, and J. H. Bellen, T., J. Littleton, J. H. Bellen, and P. Verstreken, W., T. Koh, ehti,A,Sulao,O n ace V. Haucke, and O. Shupliakov, A., Pechstein, B. Ganetzky, and L. L. Ho, M., K. O’Connor-Giles, o,T . oocu,V . ara,Y . io . vrrn . a,H,Zhou, H., Pan, E., Evergren, W., Jiao, P., Y. Wairkar, I., V. Korolchuk, W., T. Koh, ai,B,Seny .T,Psazr .E,Ro,J,Kly .B n ai,G W. G. Davis, and B. R. Kelly, J., Roos, E., K. Poskanzer, T., S. Sweeney, B., Marie, eyln u losbl ebaeuptake. membrane bulk allows but recycling P. Verstreken, h uclua oaiaino rtisi rspiasynapses. Drosophila in proteins 185 of localization subcellular the soitdwt h rsnpi yooi rjcini rspianeuromuscular Drosophila in projection cytosolic presynaptic junctions. the with associated T. endocytosis. H. McMahon, eeoeet fsnpi eil noyi eyln ehnssrvae by revealed mechanisms recycling P. endocytic neurons. Camilli, 1-null vesicle dynamin De of synaptic studies and of M. heterogeneity S. Ferguson, recycling. vesicle presynaptic aayi n al et ncsen tigpoenmtnso Drosophila. of mutants protein string cysteine in of death 263 early analysis and molecular Paralysis and Identification and larvae: integrity Drosophila (sbb). scribbler structural in behaviour for turning required Drosophila. is in zones ELKS/CAST, active al. synaptic et to of G. function Qin, homology M., with Schmidt, C., protein M. Dabauvalle, S., Buchner, junction. neuromuscular 369. Drosophila the at pools vesicle lsiiya ermsua cieznso Drosophila. of zones active neuromuscular at plasticity ebaeivgntoscae ydnmnrnsaeidcdb T-am in S GTP-gamma by induced are rings terminals. dynamin nerve by coated invaginations membrane eset Biol. Perspect. . dynamin. of Drosophila sites with interacts that growth. Chem. protein Biol. synaptic J. domain-containing during SH3 assembly multiple actin endocytic regulate Neurosci. to J. cooperate Cdc42 ebaeuo rnmte ees bevdudrrvril lcaeo membrane of blockage reversible under observed retrieval. release transmitter upon membrane h yatcvscecycle. vesicle synaptic signaling the BMP retrograde attenuate to growth. machinery synaptic endocytic during the and thickveins with oto yatcvscemmrn erea n yas development. synapse and retrieval al. 178 membrane et M. vesicle I. synaptic Robinson, endocytosis. O., control Shupliakov, vesicle J., K. Venken, and Y., development synaptic for required Neuron scaffold stabilizing 20) a10itretnsaflstepratv oet civ high-fidelity achieve to zone growth. periactive synaptic normal the and synapse. endocytosis scaffolds the Dap160/intersectin to (2004). vesicles synaptic of localization and Development transport reveals Drosophila ´lez-Gaita 273-279. , 977-980. , 309-322. , 43 .Neurosci. J. .Src.Biol. Struct. J. 193-205. , n .adJa and M. ´n, 28 Science 118 273 8316-8325. , 4 Genetics a005645. , Nature 1077-1088. , 20) nciaino ltrnhaycanihbt synaptic inhibits chain heavy clathrin of Inactivation (2008). 19108-19119. , ur Biol. Curr. 21) Copoen r ulaoso clathrin-mediated of nucleators are proteins FCHo (2010). 328 9 19) h noyi ahnr nnretriassurrounds terminals nerve in machinery endocytic The (1999). 3844-3860. , 374 Neuron 1281-1284. , 155 172 n .adSulao,O. Shupliakov, and O. ´n, 19) a10 erlseii p1 oooyand homology Eps15 neural-specific a Dap160, (1998). 18) iapaac n eomto fsnpi vesicle synaptic of reformation and Disappearance (1989). ice.Sc Trans. Soc. Biochem. cl,H. ¨ckle, 186-190. , 21) yatcvsceendocytosis. vesicle Synaptic (2012). 1161-1174. , 389-394. , Cell 9 1411-1414. , 58 507-518. , 88 rc al cd c.USA Sci. Acad. Natl. Proc. 767-776. , 19) oeo rspiaapaaatnin alpha-adaptin Drosophila of Role (1997). Neuron 21b.Asm-orltv ehiu for technique semi-correlative A (2010b). 20) el n -dependent and Cell- (2008). 21) nesci :avraieatrin actor versatile a 1: Intersectin (2010). 19) xrsino yattgi in synaptotagmin of Expression (1993). .Cl Biol. Cell J. 20) M14 aeigo synaptic of labeling 1-43 FM (2008). 20) a10itretnat sa as acts Dap160/intersectin (2004). Neuron 38 21) ehnsso short-term of Mechanisms (2010). 43 181-186. , 207-219. , 20) evu rc interacts wreck Nervous (2008). 21a.Toposo vesicles of pools Two (2010a). ehd o.Biol. Mol. Methods FPJ. HFSP 20) evu rc and wreck Nervous (2008). 49 20) p1 n Dap160 and Eps15 (2007). 182 833-844. , 1007-1016. , .Nuoc.Methods Neurosci. J. 20) rcplt a Bruchpilot, (2006). 105 4 odSrn Harb. Spring Cold 20) Abnormal (2000). 72-84. , 2175-2180. , 19) Tubular (1995). rbc,H., ¨rrbeck, .Cl Biol. Cell J. 440 Science (1994). 349- ,