nvriyMdclCne Go Center Otolaryngology, Medical of Department University InnerEarLab, Group, Neurotransmission Cochlear eateto oeua erbooy a lnkIsiueo Experimental of Institute Go Planck 37075 Max Medicine, Neurobiology, Molecular of Department nvriyo Go of University Go Center Medical University Otolaryngology, hita Vogl cells Christian hair inner cochlear vesicle in synaptic replenishment of regulation molecular Unconventional REPORT SHORT ß 638 2014 December 16 Accepted 2014; August 31 Received 5 high-resolution Recent competence. 1 fusion the full the maturation to to gain further attach tethered undergo to finally closely loosely steps and then docking first upon membrane, are membrane zone vesicles active framework, presynaptic synaptic priming. and this of docking tethering, competence In as defined fusion classically establish are vesicles that mechanisms The INTRODUCTION CAPS, Munc13, Tether, Otoferlin Priming, synapse, Ribbon WORDS: KEY Ca also (Munc13, and 13 Unc13) uncoordinated mammalian as the known of depends proteins replenishment neurons, vesicle on In cells, sound. immune and encode neuroendocrine indefatigably efficient to employ (IHCs) replenishment cells vesicle hair inner cochlear of synapses Ribbon ABSTRACT eeinmtns nta,w hwta tfri,aC a otoferlin, that CAPS and show Munc13 we isoforms. in Instead, (ABRs) mutants. CAPS well responses IHC deletion as at brainstem exocytosis and proteins IHC auditory normal CAPS as found Munc13 or and Munc13 zones active detect lacking presynaptic not did of mice we recordings Surprisingly, patch-clamp in IHC and exocytosis physiology systems with immunohistochemistry regulate auditory combining exocytosis. also by proteins IHCs for CAPS mouse in and vesicles exocytosis Munc13 whether prime tested which we Here, families, (CAPS) secretion eerhCne 8,Uiest fGo of University 889, Center Research ru,IsiuefrAdtr ersineadInraLb nvriyMedical University InnerEarLab, and Go Neuroscience Center Auditory for Institute Group, xeietlMdcn,305Go 37075 Medicine, Experimental an use IHCs in that priming vesicle conclude otoferlin. involves impaired that We machinery of priming unconventional correlate IHCs. structural which otoferlin-deficient a zone, active the be at vesicles might holding zone. tethers a short active revealed of presynaptic synapses reduction IHC the otoferlin-deficient of of tomography membrane Electron plasma IHCs, in the replenishment at and fusion clusters vesicular for crucial is that protein Germany. msrgd.e [email protected]) [email protected]; ([email protected]; correspondence for *Authors isBrose Nils etrfrNnsaeMcocp n oeua hsooyo h Brain, the of Physiology Molecular and Microscopy Nanoscale for Center of Department InnerEarLab, and Neuroscience Auditory for Institute 05 ulse yTeCmayo ilgssLd|Junlo elSine(05 2,6864doi:10.1242/jcs.162099 638–644 128, (2015) Science Cell of Journal | Ltd Biologists of Company The by Published 2015. ¨ tne,309Go 37099 ttingen, 2 eateto oeua erbooy a lnkIsiueof Institute Planck Max Neurobiology, Molecular of Department ¨ 2,4,5 tne,303Go 37073 ttingen, ¨ tne,Germany. ttingen, en-epRhee Jeong-Seop , 1, ,Bnai .Cooper H. Benjamin *, ¨ tne,Germany. ttingen, ¨ tne,305Go 37075 ttingen, ¨ ¨ tne,Germany. ttingen, tne,Germany. ttingen, 2+ dpnetatvtrpoen for proteins activator -dependent ¨ tne,309Go 37099 ttingen, 7 oeua rhtcueo Synapses of Architecture Molecular ¨ tne,309Go 37099 ttingen, ¨ tne,Germany. ttingen, 6 oisMoser Tobias , 3 6 oeua ilg of Biology Molecular erpyilg Group, Neurophysiology ¨ tne,Germany. ttingen, 2 ao Neef Jakob , ¨ ttingen, 4 Collaborative 2 -domain 1,4,5, 1 oj .Wojcik M. Sonja , n aoi Wichmann Carolin and * noriae 3(uc3 lokona n1)adCa and Unc13) as mammalian known the also to (Munc13, belonging 13 factors uncoordinated priming this employs cells, epithelial process airway and immune neuroendocrine, neurons, Ferna 99 n huh oata rmn atradvesicular and factor al., priming et a Yasunaga as 2006; act al., to et Ca thought (Roux and hearing Pangrs 1999) for (Roux 2009; cells required al., hair et is vestibular Dulon and 2006; IHCs al., et cochlear in exocytosis for ifrn egh n ubr aebe rpsdt establish to of proposed tethers been (Ferna protein competence have vesicle Instead, fusion numbers vesicular simple. in and too steps lengths distinctions been different preparatory have clear functional might such fusion and that morphological indicated of has work ultrastructural prt neoyoi tICrbo synapses. not ribbon does IHC synapses at (CNS) priming exocytosis combined system in CAPS-dependent nervous operate we and central the indicate of Munc13- data Here, machinery Our investigate conventional IHCs. determined. the in to factors that priming approaches be Munc13-like of morphological to roles and remains competence functional fusion IHCs vesicular establishing in to contribute proteins Pangrs ta. 05 h ta. 08.TeMn1 rti family protein Munc13 The 2008). al., et families Zhu (CAPS) 2005; al., secretion et for proteins (Dudenho activator dependent yassms mlymcaim fvscereplenishment vesicle multi-C of a IHC otoferlin, zone task, mechanisms involve challenging employ that active to this up For must (IHC) of second. by synapses per release cell vesicles graded Governed of hair through hundreds synapses. information inner acoustic each ribbon transmits potentials, at al., receptor release et Liu neurotransmitter 2007; al., cell et 2002). Jockusch al., several et 1999; in Varoqueaux al., 2010; neurotransmission et evoked (Augustin the arrest types of and total and loss spontaneous (RRP) dramatic complete vesicles of causes synaptic to of deletion reduction pool releasable genetic severe readily from and ranging 1999), defects, al., et Richmond conserved Speidel evolutionarily 1998; in in are al., UNC-31 CAPSs and et Brose UNC-13 and Shiratsuchi (i.e. 2001; Munc13s 2000; al., 2003). al., et al., et Betz et Koch 2001; al., 1995; isoform et family al., Augustin protein et Munc13-4 1997; CAPS al., CADPS the non-neuronal et as constitute (Ann known respectively) the (also CADPS2, CAPS2 and as and respectively) CAPS1 well whereas 3 Unc13c, (Unc13d), protein and as I-associated inhibitor Unc13b (Baiap3), angiogenesis Unc13a, brain-specific as and Munc13-3 known Munc13-2, Munc13-1, (also isoforms neuronal the includes elnsmn fteRPi ieyrt-iiigfrtonic for rate-limiting likely is RRP the of Replenishment 2+ Drosophila ´ sno o ees nIC JhsnadCamn 2010; Chapman, and (Johnson IHCs in release for -sensor dzBsaig ta. 03 isue l,20) In 2009). al., et Siksou 2013; al., et ndez-Busnadiego ˇ ic ta. 00 oxe l,20) oee,wihother which However, 2006). al., et Roux 2010; al., et ˇ fe-fie ta. 03 mge l,21;Speidel 2014; al., et Imig 2013; al., et ¨ffer-Pfeifer rvmdne l,19;Rne ta. 2001; al., et Renden 1999; al., et Aravamudan ; 2 esi Reim Kerstin , 1,4,7, * .elegans C. 2 2 ´ oanpoenta scrucial is that protein domain le Reisinger Ellen , dzBsaig ta. 2010; al., et ndez-Busnadiego ˇ ic n Uc3addCaps and dUnc13 and , ta. 02.Otoferlin 2012). al., et ˇ 3 , 2+ -

Journal of Cell Science xrse tfri Fg A.We oprn H Ca IHC comparing When 2A). abundantly (Fig. IHCs and overall otoferlin preserved the culture, was in expressed morphology week Corti a al., of After 2011). organ et al., (Nouvian et function Reisinger presynaptic 2011; of recordings patch-clamp AS/-K)o ohMn1- n uc1- (hereafter Munc-13-2 and (hereafter Ca CAPS2 Munc13-1 on and Munc13-1/2-DKO) both CAPS1 both or of the CAPS1/2-DKO) deletion investigate to genetic approach of culture effect organotypic an used We mice. K Hs(i.2;W,26.8 WT, 2H; (Fig. Munc13-1/2- and 20.9 IHCs CAPS1/2-DKO RRP in comparable kinetics DKO revealed depletion fitting and exponential size by duration ms 50 Fg ) utrdogn fCriapa omtr nlgul to analogously mature to appear Corti the of organs Cultured 2). (Fig. aiaeti prahfrsuyn Hso eiaal lethal perinatally of of IHCs analysis studying Detailed mice. for further mutant and approach culture this organotypic in otoferlin-dependence validate IHCs acquires of maturation IHCs 2006).functional al., in et P4 exhibited Roux exocytosis around 2010; Otof-KO that al., et Given (Beurg of preparations reports acute previous those using with consistent exocytosis, IHCs, reduced dramatically Munc13-1/2-DKO and epne AR)eoe ysottn ussadcik in and clicks Munc13-1 of deletion and genetic as well that bursts as Given Baiap3 CAPS2. and tone and -4 -3, CAPS1 brainstem short -2, Munc13-1, for auditory by mice (KO) recorded knockout evoked we CAPS (ABRs) and function, Munc13 responses auditory of disruption on genetic of proteins impact the assess or To Munc13 lacking factors mutants priming mouse CAPS in unaffected is Hearing DISCUSSION AND RESULTS REPORT SHORT xctsswsmntrda hne nmmrn capacitance membrane in changes ( as monitored was Exocytosis mean 2B, 306 (Fig. 292 not CAPS1/2-DKO: amplitude WT, did voltage-dependence, amplitudes we mice, maximal in (Otof-KO) differences data otoferlin-knockout detect with and Munc13-1/2-DKO WT and from CAPS1/2-DKO from currents CAPS and Munc13 presynaptic analyzed main we the function, Ca presynaptic of IHCs contributions to isoforms the Ca clarify alter not IHCs To does of factors exocytosis priming and CAPS currents or Munc13 still of Loss that numbers 1999). copy al., et protein (Augustin future provide functionality heterozygous normal require might support the will here as complete tested IHCs mice, of state knockout in effect conditional CAPS1 the on and experiments testing Munc13-1 that the of in note encoding deletion intact sound we affect suggesting However, to seem cochlea. strains not of significant does mouse disruption CAPS statistically Therefore, and mutant shown). Munc13 a not the detect (data of amplification not cochlear any did for but change function, cell outer hair evaluate to spiral emissions otoacoustic we the product Moreover, distortion S1). recorded (WT) Fig. of material wild-type supplementary to potential 1; compared when (Fig. action littermates mutants the compound of any the in ganglion, reporting of ABR the alterations I, of latencies observe or wave amplitudes not in did changes nor We thresholds ABR genes. mice these from ABRs for recorded heterozygous we lethality, perinatal in results CAPS1 eoaiaino ayn uain Fg DG.Atog the Although 2D–G). (Fig. durations D varying of depolarization D C C 2+ m nvivo in m 6 6 auswr nitnusal ewe T CAPS1/2-DKO WT, between indistinguishable were values nrsos otemxmlCa maximal the to response in ) urnsadeoyoi ntersetv eeinmutant deletion respective the in exocytosis and currents . F uc312DO 31.9 Munc13-1/2-DKO, fF; 5.4 s.e.m., iuto Sboize l,18)adaesial for suitable are and 1982) al., et (Sobkowicz situation nvivo in P . .5btenalgop)o ieis(i.2C). (Fig. kinetics or groups) all between 0.05 Buge l,21) u idnsidct a indicate findings our 2010), al., et (Beurg 6 0p,Mn1-/-K:286 Munc13-1/2-DKO: pA, 30 2+ die xctssi IHCs in exocytosis -driven 6 5p;Oo-O 291 Otof-KO, pA; 25 D C 6 6 m . F CAPS1/2-DKO, fF; 4.2 . fF; 5.5 ausfrsiuio 2– of stimuli for values 2+ nlxeiie by elicited influx P . .5between 0.05 6 6 nvitro in 0pA; 20 1pA, 31 2+ 2+ n hne nCa detect in not did changes IHC but Baiap3, presynaptic and any Munc13-4 tested lacking we mice in Otof-KO Moreover, function of analysis. exocytosis reduced such strongly prohibited the whereas groups), all aeilFg 1) ecnld htMn1-iepriming Munc13-like that function. conclude presynaptic IHC for We dispensable are S1B). factors Fig. material rmciknccla xrcs(taa n useh 2010), Hudspeth, and (Uthaiah absent extracts is protein cochlear Munc13-1 chicken that from indicated have factors priming reports Munc13-like Previous lack apparently IHCs Cochlear C (MHD; 2000). domain al., et homology Koch Munc13 from the adapted of conservation CAPS the and highlighting Munc13 in unaffected mutants. remain deletion thresholds Hearing 1. Fig. nml.ARwvfrs(–)i epnet 0d lc tml r shown are stimuli click dB 80 CAPS1 to (F) response for in (I–V) waveforms ABR animals. ausaesono h figures. the on shown are values B hehlsi B Munc13-1 (B) in thresholds ABR D Munc13-3 (D) ora fCl cec 21)18 3–4 doi:10.1242/jcs.162099 638–644 128, (2015) Science Cell of Journal 2 +/ / 2 2 n E CAPS1 (E) and CAPS2 A oanoeve fMn1-iepoenisoforms protein Munc13-like of overview Domain (A) 2+ 2 / 2 urnso xctss(supplementary exocytosis or currents n G Munc13-1 (G) and +/ +/ 1 2 2 ,C n Munc13-4 and CAPS2 1 oan C domain; 2 / 2 +/ ieadaemthdWT age-matched and mice 2 2 ,C 2 ierespectively. mice / 2 2 C Munc13-2 (C) , oan Comparable domain. 2 n / 2 639 ,

Journal of Cell Science HR REPORT SHORT emnl fefrn lvccla ern,a vdn from evident presynaptic as 640 to neurons, restricted including olivocochlear were proteins, efferent Munc13-2 of tested not terminals IHCs. all detect but within not of our from Munc13-1 immunofluorescence did immunoreactivities we with CAPS Corti of function, Rather, or role Consistent of IHC Munc13 functional presynaptic 3). organs specific a in (Fig. proteins against dissected mice these arguing acutely data WT established. (Cooper on electrophysiological isoforms P16–P17 be CAPS 2012) extensively and hearing to al., Munc13 with remain all et immunostainings for cochlea antibodies performed tested the and we Munc13 in remaining Therefore, the isoforms of patterns CAPS expression the however, h ointa hi neatn atesnuoa soluble neuronal partners interacting their with line that in is that notion finding operate a to zones. function proteins, the seem active a CAPS IHCs IHC or that presynaptic from plays establish Munc13s release could without we isoform vesicular of study, in present this role the – In analysis that any if S1B), – we our minor Fig. IHCs, in material on expression (supplementary Munc13-4 based is there conclude, that from we absent exclude Although was antibodies. cannot that available labeling currently a with (i.e. tissue) for knockout Corti in immunolabeling of controls specific organs positive a in find internal Munc13-4 not as did served We experiments. which these 2), and synapsin marker 1 vesicle (isoforms synaptic neuronal the with colocalization ora fCl cec 21)18 3–4 doi:10.1242/jcs.162099 638–644 128, (2015) Science Cell of Journal tfK essWT. versus Otof-KO ie sqatfe nH l eoye,aatfo Otof-KO, from * apart amplitudes. genotypes, RRP All comparable H. showed in quantified as sizes rmn atrmtnsadOo-OIC tDV–.Voltage- DIV7–8. at Ca IHCs and Otof-KO dependence and mutants factor priming Hs la rohasotrhi el,OC B Whole-cell (B) OHC. cells, hair Ca indicate outer arrowheads arrowheads filled clear (red); IHCs; F-actin and (green) otoferlin days 7 after confocal culture a organotypic of animals. projection Otof-KO Maximum in (A) impaired but strongly Munc13-1/2-DKO is and exocytosis CAPS1/2-DKO in conserved are Ca 2. Fig. fiinybtenMn1-iemtnsadW Hs G Initial (G) IHCs. WT D and mutants Munc13-like between efficiency eoye D Exocytic (D) genotype. eoaiain n orsodn Ca corresponding and depolarizations ucino Ca of function a eoaiaint h aiu Ca maximum the to depolarization Mean (C) genotypes. K Hs nOo-O xctssi rmtclyreduced. Ca dramatically maximum is Mean exocytosis (E) Otof-KO, In Munc13-1/2- IHCs. and DKO CAPS1/2-DKO WT, in exocytosis comparable epciegop o 0m eoaiain (F) depolarization. ms 50 a for groups respective C 2+ m eefte iha xoeta ucint siaeRRP estimate to function exponential an with fitted were urn–otg eainhp rmclue Munc13-like cultured from relationships current–voltage 2+ urns R ieadssandexocytosis sustained and size RRP currents, 2+ urn hresoscmaal release comparable shows charge current 2+ n 6 urn mltdsaertie cosall across retained are amplitudes current ausaesono h figures. the on shown are values ...sml rcso 0ms 50 a of traces sample s.e.m. 2+ D C urn n corresponding and current m nrsos ovrigstep varying to response in nvitro in 2+ urn oeta o each for potential current 2+ z DV muotie for immunostained WT (DIV) a from taken -stack P urn nerl show integrals current , .5 *** 0.05; D C P m , D lte as plotted C .0 for 0.001 m fthe of

Journal of Cell Science oivsiaevsclrttesa cieznso Tand WT of zones active at tethers vesicular synapses IHC of investigate tomography electron to used we Therefore, 2010). HR REPORT SHORT mlctda nutatutrlcreaeo eiua fusion vesicular of been has correlate which ultrastructural (Ferna tethering, an competence might vesicle as site in release implicated labelings involvement the immunogold at an of with enrichment observed base indicate found Otoferlin the be shown). and at also not an these membrane (data could otoferlin zone recognizes that In active ribbon of that the the zones. in antibody epitope clustering active otoferlin an C-terminal zone neuronal employed and active intraluminal at Munc13 we the to factors at experiments, analogous priming otoferlin localization a of CAPS 4A), enrichment (Fig. an membrane confocal mechanism revealed Our the replenishment. data investigate vesicle facilitates further otoferlin to classical which the by aimed without we operate factors, to seems priming exocytosis IHC that and Given membranes formation zone tether active vesicular IHC regulates in enriched is Otoferlin al., (Pangrs otoferlin et and is (Frank 2011) bassoon al., IHCs and/or et Snellman of ribbon 2010; synaptic machinery and the zones priming involves active likely neuronal the conventional from that distinct our molecularly propose on Based 2011). we al., et (Nouvian findings, IHCs from absent be to appear efferent (blue (SNAREs) olivocochlear IHC receptors N-ethylmaleimide-sensitive-factor counterstain attachment typical to a simplicity. 2) of for illustration and complex schematic 1 synaptic a (isoforms single shows synapsin a D and in showing marker inset innervation IHC The neuronal clarity. as (red) for used efferent alongside was and presented (calb.) (white) are Calbindin channels CAPS2. Individual (D) terminals. and factors. presynaptic CAPS1 priming (C) Munc13-like Munc13-3, lack (B) apparently IHCs 3. Fig. ´ dzBsaig ta. 00 rn tal., et Frank 2010; al., et ndez-Busnadiego ˇ ic ta. 2010). al., et ˇ igecnoa lnso 1–1 Togn fCriimnsandfr()Munc13-1 (A) for immunostained Corti of organs WT P16–P17 of planes confocal Single tfri-eiin Hs ntnaeul rznfollowing frozen K high instantaneously with stimulation IHCs, otoferlin-deficient n ciezn ebae sti ouainhsbe rpsdto proposed tethers (Ferna been vesicles has readily-releasable filamentous population represent this as on membrane, zone active analysis and density presynaptic our the to vesicles focused membrane-proximal connecting We 4B–G). (Fig. twl ecuilt dniyadcaatrz neato atesof are partners interaction characterize and membranes identify to plasma crucial be Additionally, will studies. and it future in investigated vesicular be to remains to otoferlininvolved, of interactions localized homophilic so, synaptosomes molecules if hypertonic and, in constituent tether or tethers a neurotoxins short clostridial deficit, (Ferna exocytosis of sucrose with the reduction treatment to related the following the directly that to speculate is to tethers analogous tempting short is of It 4E–H). loss Otof- nm, (Fig. at 10 zones lengths of active than tether KO shorter average reduction, tethers increased significantly of significant in fraction resulting statistically the in a two-thirds, roughly detected shown). we membrane- not of (data However, number vesicles the ribbon-associated in neuronal overall (Roux difference and described no proximal without was previously there As operate 2006), al., 2011). et ribbons al., et IHC (Nouvian SNAREs that suggesting report tethers detect (Ferna to failed we Interestingly, , 2010). al., et Frank 2010; m huh ocmrs sebe NR complexes SNARE assembled comprise to thought nm, 5 ´ dzBsaig ta. 00,cnitn ihaprevious a with consistent 2010), al., et ndez-Busnadiego ora fCl cec 21)18 3–4 doi:10.1242/jcs.162099 638–644 128, (2015) Science Cell of Journal ´ dzBsaig ta. 00.Wehrooelnis otoferlin Whether 2010). al., et ndez-Busnadiego + oaayeogigsnpi activity synaptic ongoing analyze to ´ dzBsaig tal., et ndez-Busnadiego ihafferent with ) 641

Journal of Cell Science HR REPORT SHORT uc3adCP rtisapa ob betfo IHC from absent be to 642 CNS appear (3) and proteins glutamatergic abolished, CAPS completely at and or impaired release Munc13 strongly is whose and is synapses Munc13-1/2-DKO hearing mice in (1) (2) normal factors, that is CAPS1/2-DKO priming time exocytosis Munc13-like presynaptic various first of IHC the absence the for CAPS in show and intact Munc13 we IHC for factors, mutants cochlear priming deletion at Using synapses. machinery ribbon priming molecular unconventional fusion vesicular establishing to contribute competence. might that otoferlin nsmay u td rvdseiec o an for evidence provides study our summary, In ihyefcetrpeiheto yatcvsce nIHCs. in vesicles synaptic of the for replenishment mechanism efficient candidate highly a providing membranes, zone active rsnpi ciezns nta 4,ooelnapast be to ( appears tethers otoferlin short (4), form Instead to required zones. active presynaptic 05,Mn1-/-K Agsi ta. 99 aouaxe al., et Varoqueaux al., 1999; et al., Speidel et 2007; al., (Augustin et Munc13-1/2-DKO Wild-type (Jockusch Saxony. 2005), Lower CAPS1/2-DKO of office (WT), welfare C57Bl/6 animal and guidelines the care by animal approved national were to conformed experiments animal ABRs All and culture organotypic Animals, METHODS AND MATERIALS h figures. the uli h oe rai rsne saconfocal IHC a indicate as presented Asterisks is (arrowheads). area the boxed zone The of active nuclei. apposition the apparent at showing proteins CtBP2, two marker and ribbon membranes synaptic zone tethering. the active vesicular IHC in in role a enriched plays is Otoferlin 4. Fig. H uuaiepoaiiydniyposo ehrlntsrwdata. raw lengths tether and of binned) plots * nm density mean (5 probability increased histograms cumulative an Normalized (H) shift and (G) significant (E), (F). highly samples length a Otof-KO tether to in leads tethers tethers longer short genotypes towards of both in reduction vesicles the synaptic (D), Despite tethered of arrowhead). numbers (clear comparable tether plasma the filamentous the a to through connected (PM) vesicles membrane of images (SVs). Representative vesicles (C) presynaptic synaptic magenta, membrane-associated membrane; yellow, zone density; active Blue, models. rendered oorpi etoso B)W n Bi tfK ibnsnpe after synapses ribbon K Otof-KO high (Bii) with and stimulation WT Representative (Bi) segregated (B) of is ribbon. sections that CtBP2-labeled tomographic plane the a containing in those otoferlin from of clustering the illustrating ribbon, P , .5 *** 0.05; ora fCl cec 21)18 3–4 doi:10.1242/jcs.162099 638–644 128, (2015) Science Cell of Journal P , .0 o tfK essWT. versus Otof-KO for 0.001 + oeoemmrn unvrwt corresponding with turnover membrane evoke to , A muotiigfrooelnand otoferlin for Immunostaining (A) 0n)btenvsce and vesicles between nm) 10 n ausaesonon shown are values z sako h indicated the of -stack

Journal of Cell Science E-l(0,4aioprdn 1) MgCl (10), (130), 4-amino-pyridine Cs-gluconate mM): (10), (in TEA-Cl containing solution intracellular Cs-based 02,Mn1--O(uutne l,20)adCAPS1 and 2001) al., et (Augustin Munc13-3-KO 2002), uato Tmc fete e nHPSbfee HBSS HEPES-buffered in sex 10 either and 18–19 of day fungizone embryonic ng/ml 2011). mice 250 from al., with WT dissected et supplemented were as Reisinger or Corti Corti 2011; of mutant of organ al., the organs et of Briefly, (Nouvian cultures either organotypic previously of prepare mice described to 2011) used al., were et Reisinger sex (Otof-KO; otoferlin-KO and 2002) REPORT SHORT otd10cpe ehgisadps-tie ih4 uranyl-acetate 4% with post-stained and formvar- grids on 250-nm described mesh (Leica). collected copper as electron-tomography, were 100 HPM100 Leica) For (Leica) coated ultramicrotome; a 2006). AFS2 E al., (Ultracut an with sections et in frozen (Rostaing performed were previously was 200- solution specimens substitution in stimulation the Freeze placed with and were filled P14) Aldrich) carriers (all specimen mice (Pangrs A from Otof-KO Corti type two of aluminium and organs apical-turn WT explanted three freezing, high-pressure reconstruction For tomographic and microscopy Electron to conjugated stereocilia. antibodies containing anti-IgG AlexaFluor secondary mouse detected and anti-CAPS2 AlexaFluor Systems), species-specific (Swant) (Abcam/Synaptic anti-CAPS1, 2 antibodies D28k by and 2005), anti-otoferlin 1 anti-calbindin rabbit synapsin al., mouse or both against Systems), et antibody (Cooper (Synaptic pig Varoqueaux guinea antibodies Systems), 2012; (Synaptic anti-Munc13-3 al., anti-Munc13-1, isoform), et rabbit anti-ubMunc13-2 Munc13-2 isoform), polyclonal S1A) (ubiquitous Munc13-2 immunolabellings: Fig. (brain-specific material for anti-bMunc13-2 (supplementary used antibodies were primary following The Antibodies 63 NA 1.4 laser-scanning a SP5 or with SP2 al., Leica microscopes et using (Neef confocal imaged previously were described Specimens as 2009). preparations whole-mount organs apical Corti dissected of acutely on performed was Immunohistochemistry microscopy confocal and Immunohistochemistry 22–25 at done were experiments All kinetics. release RRP and currents cultures); for 10 KCl preparations, (103), acute NaCl for mM): (2 (in Otof-KO) of composed MgCl CAPS1/2-DKO; (2.8), solution (Munc13-1/2-DKO; extracellular an Corti cultured from using of IHCs or organs Baiap3-KO) apical acutely (Munc13-4-KO; either mice from P14 IHCs dissected on performed were recordings Perforated-patch IHCs of Patch-clamp isine)adicbtdi MMF2wt %FSfr78days 7–8 for FBS 5% characterization. with electrophysiological DMEM/F12 to in prior incubated and Biosciences) eela-utatdwt /0pooo AmtogadBezanilla, and (Armstrong protocol of exceeding currents p/10 leak with a Lindau-Neher Currents IHCs 1974); 2000). with Beutner, the and leak-subtracted Moser using were 1988; Neher, performed and (Lindau were technique measurements electronics). (HEKA software Capacitance PatchMaster with amplifier EPC10 an using (250 , B hehlsa ecie rvosy(efe l,2009). al., et (Neef previously Jackson testing described for alongside mice; as mice WT thresholds used (Jinx C57Bl/6 ABR were old) Munc13-4-KO weeks 2013) (3–10 age-matched al., as or et littermates well (Wojcik as Baiap3-KO and 2007) Laboratories) al., et (Jockusch SgaAdih,te lcdo CellTak on placed then (Sigma-Aldrich), betv,rsetvl.Iae eetrsoddfrbackground for 2012). al., thresholded et (Schneider were ImageJ using deconvolved Images and subtraction respectively. objective, acltdwt h grPoLPcluao n orce offline. corrected and calculator LJP Pro Igor the with calculated 9 Oml H71fehysplmne ihapoeii B amphotericin with supplemented freshly 7.1 pH mOsm/l, 290 Munc13-1 2 7m eedsaddfo nlss iudjnto oetaswere potentials Liquid-junction analysis. from discarded were mV 87 m ˇ /l.W mlyda lvtd[Ca elevated an employed We g/ml). ic ta. 00.Tp iswr ipdi eaeee(Sigma- hexadecene in dipped were lids B Type 2010). al., et ˇ H H 2 +/ 6 Lf ehoois a sdt ae actin- label to used was technologies) (Life 568 1,HPS(0,TAC 3) -lcs 1.)adCaCl and (11.2) D-glucose (35), TEA-Cl (10), HEPES (1), 2 8,58ad67(ietcnlge) Phalloidin– technologies). (Life 647 and 568 488, Agsi ta. 99,Mn1--O(aouaxe al., et (Varoqueaux Munc13-2-KO 1999), al., et (Augustin 2 0p ttehligpotential holding the at pA 50 , 2+ 6 TM 2 0 Oml H72ada and 7.2 pH mOsm/l, 300 nodrt nac Ca enhance to order in ] 00)adHPS(10); HEPES and (0.05) cae oesis(BD coverslips -coated r100 or m /lPncli G Penicillin g/ml 6 +/ oil-immersion 2 -CAPS2-KO m ˚ 2+ m C 2 uutn . oemn,C,Su C., Rosenmund, I., Augustin, F. Bezanilla, and M. C. Armstrong, uutn . ot,S,Rcmn,M,Kezcmr .A,Su A., H. Kretzschmar, M., Rickmann, S., Korte, I., Augustin, rs,N,Hfan . aa .adSu and Y. Hata, K., Hofmann, N., R., Brose, Schneggenburger, Y., Bouleau, V., S., Safieddine, Scheuss, N., J.-S., Michalski, Rhee, M., Beurg, U., Ashery, J., H. Junge, P., Thakur, A., Betz, K. Broadie, and K. C. Rodesch, S., W. Davis, T., Fergestad, B., Aravamudan, E 10tasiso lcrnmcocp t20k ttl angles tilt at kV JEOL 200 a at at acquired microscope from were electron series were ranging tilt transmission particles double 2100 or gold Single JEM 10-nm grid. the Subsequently, to applied lead-citrate. Reynold’s and ehia sitne oevr ewudlk oepesorgaiuet Nicola to gratitude manuscript. our the express Pangrs to Tina like and would Strenzke we Christian Moreover, thank assistance. We technical mice. Munc13-4-KO providing for Ru Rettig Jens thank We tether Acknowledgements (i.e. data fractional of comparison a length), For groups test); multiple significant. of Kolmogorov–Smirnov statistically data distributed by normally (assessed on mean performed were as Tukey presented are Data analysis Statistical n,K,Kwlhk .A,Lyt .M n atn .F J. F. T. Martin, and M. K. Loyet, A., J. Kowalchyk, K., Ann, References at http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.162099/-/DC1 online available material Supplementary material N.B.]. Supplementary to and A4 J.R. T.M, to to B1 A2 and [projects C.W. 889 (DFG) to SFB Foundation A7 Center Research E.R., Center Research German Medical Collaborative the University the of the through grants of and grant C.V. intramural to an Goettingen by supported was work This Funding manuscript. the prepared C.W. T.M. and and J.N. C.W. C.V., C.V., preparation. data. provided manuscript the and the analyzed mutagenesis into mouse input performed and N.B. discussion tomography. and EM K.R. C.V. S.M.W., performed electrophysiology. C.W. E.R., performed immunohistochemistry, C.V. performed study. B.H.C. the and designed T.M. and C.W. C.V., contributions Author interests. financial or competing no declare authors The interests Competing md) ehrlntswr nlzdi Dfo oorm using ( tomograms starting from of 3D in determination IMOD analyzed by the (http://bio3d.colorado.edu/ were 3dmod using ImageJ lengths using Tether generated rendered imod/). and were etomo Tomograms package software. Serial-EM oriae nvrulscin n acltn h egh ihthe with lengths the calculating and formula: sections following virtual in coordinates once otepeyatcdniyadatv oemmrn were membrane zone active analysis. and the density in presynaptic considered the to connected dgr tfnTo,Snr ek n hitaeSne-ria o expert for Senger-Freitag Christiane and Gerke Sandra Thom, Stefan ¨diger, h pnn n lsn fteatvto ae fteN channels. Na the of gates activation the of closing Physiol. and opening the seta o uincmeec fguaaegcsnpi vesicles. synaptic glutamatergic of 457-461. competence fusion for essential Neurosci. ooouso anradtseeasuc1 eedfn oe aiyo C2- of family novel define gene proteins. unc-13 domain elegans Caenorhabditis of homologues D. Dulon, and cells. hair auditory C. in otoferlin Petit, and synaptotagmins R., E. priming. Chapman, vesicle synaptic in RIM1 and Munc13-1 30 proteins N. Brose, zone and active J. in Rettig, learning C., Rosenmund, motor and transmission synaptic mice. cerebellar N. regulates Brose, Munc13-3 and W. J. Herms, ess is UNC-13 Drosophila (1999). idn rti CP)rela (CAPS) protein binding exocytosis. 183-196. , .Neurosci. J. 63 x ora fCl cec 21)18 3–4 doi:10.1242/jcs.162099 638–644 128, (2015) Science Cell of Journal 2 2 533-552. , 965-971. , ts a used. was -test .Bo.Chem. Biol. J. 2 .Bo.Chem. Biol. J. 62– 21 ! 10-17. , 2 [( ˇic x 55 ˇ 2 Vla o uprigtesuyadciial reading critically and study the supporting for -Vilfan 2 272 ˚ x 20) h eeelmseii uc3isoform Munc13 cerebellum-specific The (2001). o+55–+62 to 1 6 19637-19640. , hf .C n rs,N. Brose, and C. T. dhof, ¨ e oUC3 eurdfrCa for required UNC-31 to ted ) 2 270 ...OewyAOAwt post-hoc with ANOVA One-way s.e.m. +( y 17) hremvmn soitdwith associated movement Charge (1974). 25273-25280. , 2 2 nilfrsnpi transmission. synaptic for ential y 1 ) 20) ucinlitrcino the of interaction Functional (2001). 2 hf .C. T. dhof, ¨ ˚ +( x 21) oto feoyoi by exocytosis of Control (2010). 1 z ih1 with y 2 P 1 2 .Neurosci. J. z # 1 z n nig( ending and ) .5wsacpe as accepted was 0.05 1 ) 2 .Snpi vesicles Synaptic ]. ˚ nrmnsusing increments 19) Mammalian (1995). 19) uc31is Munc13-1 (1999). 19) oe Ca Novel (1997). 30 13281-13290. , hf .C., T. dhof, ¨ 2+ Nature -activated .Gen. J. x Neuron 2 y 643 400 Nat. 2 z 2+ 2 ) - ,

Journal of Cell Science i,Y,Shra . dlan . at,U,Re,J,Hf . rn,D,Brose, D., Bruns, D., Hof, J., Rhee, U., Matti, L., Edelmann, C., Schirra, Y., Liu, E. Neher, and M. Lindau, ef . er,A,Blnia .V,Myr .C,Ree,D,Geg .G., R. Gregg, D., Riedel, C., A. Meyer, V., A. Bulankina, A., Gehrt, J., Neef, D. Beutner, and T. Moser, N. Brose, and K. Hofmann, H., Koch, ono,C .adCamn .R. E. Chapman, and P. C. Johnson, Rhee, F., Varoqueaux, B., J. Sørensen, A., Sigler, D., Speidel, J., W. Jockusch, mg . i,S-. rne,S,Aaclo . oemn,C,Su C., Rosenmund, M., Arancillo, S., Krinner, S.-W., Min, C., Imig, Ferna C. Petit, and M. S. Jones, S., Safieddine, D., Dulon, Dudenho Ammermu M., Hemmerlein, B., Cooper, REPORT SHORT 644 Ferna Pangrs Pangrs E., Reisinger, V., A. Bulankina, J., Neef, R., Nouvian, rn,T,Rtefr,M . teze . ef . Pangrs A., Neef, N., Strenzke, A., M. Rutherford, T., Frank, Pangrs esne,E,Bee,C,Ne,J,Ni,R,Rue,K,Blnia . 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