oeua eiieadSaeKyLbrtr fBoebaeEngineering, China. Biomembrane 100871, of Beijing Laboratory University, Key Peking State and Medicine Molecular ipyis hns cdm fSine,Biig100,China. 100101, Beijing Sciences, of Academy Chinese Biophysics, Ato o orsodne([email protected]) correspondence China. for 100053, *Author Beijing Disorders, Brain for Institute Beijing Disease, Alzheimer’s eeomna ilg,CieeAaeyo cecs ejn 011 China. 100101, Beijing Sciences, of Academy Chinese Biology, Developmental & hnu Hao Zhenhua cells in chromaffin vesicles adrenal dense-core muted-deficient large of maturation Impaired ARTICLE RESEARCH ß eevd6Ags 04 cetd2Fbur 2015 February 2 Accepted 2014; August 6 Received 2 (Yu for biogenesis LDCV lysosomes 1 during into Castle, important 2011). mis-sorting and al., also et (Arvan cargo is retention of for degradation LDCVs: sorting Prevention into Two and sorting and 1998). 2001). entry cargo al., mature for for et proposed into sorting Tooze been 2006; assembly have al., cargo hypotheses et and (Kim proper are players vesicles fully machineries ensure condensed not molecular sorting to are biogenesis, the important LDCVs LDCV of However, During biogenesis understood. 2013). the governing (LRO) Li, organelle mechanisms cells, lysosome-related and of type secretory (Wei a as specialized LDCVs characterized cells. are in non-neuroendocrine of and the secretion neuroendocrine neuropeptides for at including organelles and formed key are are hormones which (TGN), network (LDCVs), trans-Golgi vesicles dense-core Large INTRODUCTION protein, Muted syndrome BLOC-1, Hermansky–Pudlak vesicle, CgA, core dense Large WORDS: LDCVs. KEY of were biogenesis the frequency during CgA the release in of involved export is vesicle selective protein muted the the that suggest studies and Our reduced. pool releasable In readily docking. impairs and which LDCVs, maturation immature of LDCV out molecule this export to failure mouse LDCVs in that enlarged a found were we is Here, syndrome. (BLOC-1), Hermansky–Pudlak of complex-1 model organelles lysosome-related of and The as unknown. known hormones the (also largely on mutation null is spontaneous a of carries pathway which mouse, biogenesis secretion this sorting granin a for to machinery is the the family but biogenesis, granin LDCV in The in force driving cells. secretory lysosome-related specialized involved in of neuropeptides type is a (LDCV), organelle, vesicle dense-core large The ABSTRACT mrooy hniMdclUiest,Tiun000,China. 030001, Taiyuan University, Medical Shanxi Embryology, ncdw el.TeicesdCAin CgA increased in The increased cells. was knockdown CHGA) as known also e Li Wei nvriyo hns cdm fSine,Biig103,China. 100039, Beijing Sciences, of Academy Genetics Chinese of of Institute University Biology, Developmental Molecular of Laboratory Key State 05 ulse yTeCmayo ilgssLd|Junlo elSine(05 2,16–34doi:10.1242/jcs.161414 1365–1374 128, (2015) Science Cell of Journal | Ltd Biologists of Company The by Published 2015. 1,6, * Bloc1s5 mu 1,2 iiWei Lisi , dea hoafncls hoornnA(CgA, A Chromogranin cells. chromaffin adrenal ,wihecdsasbnto h biogenesis the of subunit a encodes which ), 3 ai Feng Yaqin , mu 4 eateto itlg and Histology of Department hoafncls h ieof size the cells, chromaffin mu mu iewslkl u a due likely was mice deasadmuted- and adrenals 1,4 ioe Chen Xiaowei , 5 nttt of Institute Muted mu 6 etrof Center 3 nttt of Institute mutant gene 3 e Du Wen , h aeptwyi euaigedsmltafcig(Angers trafficking endosomal regulating in in act might pathway 2006). and physically al., same interact et the complex Grabner an transmitter HOPS 2010; the of and al., and amount et as AP-3 number the (Asensio known vesicle) (i.e. decreased per size also released quantal size, (SgII, releasing enlarged in II increase exhibit secretogranin also and (CgA, and chromaffin A SCG2), CHGA) chromogranin AP-3-deficient as granins, In less known contain LDCVs. LDCVs unknown. immature cells, granin largely in into the involved is likely but is sorting LDCVs complex 2001), (AP-3) into al., protein-3 adaptor sorting et The granin (Kim biogenesis for LDCV machinery in role important ed ovral hntpso Rs oprsno the of Comparison LROs. on subunit 2014). BLOC-1 phenotypes al., et different variable Zhang of 2005; al., to deficiency al., et the leads et that (Cai Tian suggests 2014; autophagy This al., impaired et exhibit Scott 2010; and are lethal the mice BLOS1-knockout as embryonic and such snapin- whereas BLOC-1 mice, deficiency) of with mice mutants compared deficiency), LROs LRO mouse KXD1-knockout (dysbindin in HPS by defects The mild typical characterized exhibit 2013). other 2012) is Li, al., which et and (Yang humans, (Wei and dysfunction al., mice et in syndrome Peter Hermansky–Pudlak (John (HPS) causes maturation deficiency endosomal BLOC-1 and see 2013). 2013) review, Current a Li, (for 2012). and lysosomes on Wei al., to focused et endosomes mainly from are Yang trafficking BLOC-1 2013; cargo of (Hayes al., functions BLOC-1 et the of of Peter understanding subunit John additional 2011; an al., KXD1 2004). et as Dell’Angelica, identified and been Starcevic BLOC1S3) 2003; has as al., et Falco known Li 2003; (also (also 2004; al., BLOS2 BLOS3 snapin, et GCN5L1), and (Ciciotte or BLOC1S2) BLOC1S5, respectively), BLOC1S1 as as as BLOC1S4, known known known (also and BLOS1 (also DTNBP1 cappuccino BLOC1S6, dysbindin, the limited. very control pallidin, are proteins complexes LDCV of trafficking LDCVs processes export these and immature out how import underlying molecules into the of likely unnecessary However, export). cargoes mechanisms of are (selective sorting LDCVs of BLOC-1 immature the These of sorting and or 2008). import) HOPS the al., (selective et AP-3, in (Chen that involved size suggest quantal findings of increased secretion reduced biogenesis to and leading of size, events vesicle subunit in increased in and [a reduced cells number are dysbindin chromaffin (BLOC-1)] complex-1 adrenal in (Asensio organelles lysosome-related in cells deficient 2008). LDCVs AP-3-deficient mice the al., in to from Similarly, et leads 2013). as cells al., Wilkin LDCVs PC12 et 1999; in of subunit) defects al., HOPS similar (a et VPS41 Rehling of Knockdown 2009; Merz, and rvossuishv ugse httegai aiypasan plays family granin the that suggested have studies Previous h LC1cmlxcnit fegtsbnt:muted, subunits: eight of consists complex BLOC-1 The 5 igMa Jing , 1 ha Zhou Zhuan , mu ´ mtddfcec)and deficiency) (muted n-Pe ´ e ta. 02 wn tal., et Gwynn 2002; al., et rez 3 iny Chen Liangyi , pa (pallidin 3 1365 and sdy

Journal of Cell Science u io td,w bevdthat observed In we 2002). study, al., as pilot et such our (Zhang LROs granules several platelet-dense of and biogenesis melanosomes the affects and protein muted the on mutants of mouse involvement their BLOC-1 in biogenesis. different mechanisms LRO underlying in the defects reveal might LRO of features ARTICLE RESEARCH 1366 in affected are LDCVs whether investigate in To LDCVs of changes Morphological RESULTS the of export biogenesis. Tian the LDCV in 2009; involved during is mutants, al., CgA protein mouse granin muted et HPS the additional (Pan that with propose mice comparison we In snapin-knockout 2005). al., or et 2008) al., to compared et LDCVs in changes morphological of ntecnrlmc Fg A.Terudgaua iiswere limits granular round The in condensed 1A). were cores (Fig. dense and mice visible control the in of ultrastructure gross adrenal ( The from controls LDCVs of of changes gland morphological the analyzed we The mu Muted uatmue hc are pnaeu mutation spontaneous a carries which mouse, mutant ee( gene mu Bloc1s5 + and /+) mu ,aoihsteepeso fthe of expression the abolishes ), hoafnclswssmlrt that to similar was cells chromaffin mu mu ieb lcrnmicroscopy. electron by mice iehdadfeetprofile different a had mice mu mu hoafncells chromaffin hoafncls In cells. chromaffin mu sdy ie( mice ie(Chen mice mu/mu ), PreaGmsadSrdbr,20) nthe in 2001), Stridsberg, and islet type (Portela-Gomes in another in size LDCV core dense of and size vesicle enlarged observed by increased mu was diameter vesicle to compared diameter in ftemtdpoenlkl fet DVmrhlg na in morphology LDCV affects likely protein loss mechanism. muted conserved that suggest the results These of S1). Fig. material (supplementary by in enlarged LDCVs significantly was of (diameter) size (diameter) core size dense the was cells of chromaffin increase included. average were core round well-preservedThe electron-dense a an with and halo granules a round membrane, no square all analysis, per was size vesicles vesicle There For and of control genotype. number between total each micrometer the for in difference animals cells selected significant the different randomly examined of two then sections in We from LDCVs 1A). of (Fig. size vesicles of and more whole number occupied the cores in dense space the in and the However, spacious core. less dense-core dense were packaged the halos the around loosely halo a spacious had a and granules many cells, control mu iecmae ihcnrlmc Fg E.Smlry we Similarly, 1E). (Fig. mice control with compared mice hoafncls(i.1) ec,tertoo es core dense of ratio the Hence, 1D). (Fig. cells chromaffin ora fCl cec 21)18 3517 doi:10.1242/jcs.161414 1365–1374 128, (2015) Science Cell of Journal 24vsce)ta ncnrlmc (0.594 *** mice vesicles). control 7664 in than vesicles) 6244 in higher diameter:vesicle was core dense diameter of frequency ratio a The as histogram. presented is diameter LDCV *** (135.52 vesicles). mice control in that (159.43 in of increased diameter cores average The electron-dense histogram. presented frequency is a diameter as core electron-dense The (D) cl as 0.5 bars: Scale mu 24vsce)cmae ihta ncnrlmice control in that (237.30 with compared vesicles) 6244 the of in diameter increased mean LDCVs as The presented histogram. is frequency diameter a LDCV The (C) significant. ifrnei h oa ubro eilsin vesicles 5.34 of (con: number total the in difference from sections heterozygous gland adrenal of micrographs in cells. LDCVs of chromaffin pathology Ultrastructural 1. Fig. vesicles/ a cls hc oti g n glucagon and CgA contain which -cells, , ( %(i.1) utemr,teelectron- the Furthermore, 1C). (Fig. 6% mu 6 6 ih)ml iea h g f8weeks. 8 of age the at mice male right) , m .1n,64 eils oprdwith compared vesicles) 6244 nm, 0.71 .7n,76 eils.*** vesicles). 7664 nm, 0.87 m 6 2 .6vesicles/ 0.36 P .Rslsaemean are Results ). , mu mu .0.()Tednecr diameter/ core dense The (E) 0.001. m .()Teewsn significant no was There (B) m. homozygous and left) (con, /+ P A ersnaieelectron Representative (A) , hoafncls(i.1B). (Fig. cells chromaffin mu 0.001. mu ie(251.25 mice m m 6 ie(0.645 mice 2 .0n,7664 nm, 0.60 ; mu 6 5.00 : ...NS,not N.S., s.e.m. mu P 6 mice , 6 .8nm, 0.88 6 mu 0.001. 0.002, , mu 0.24 6 , mu 0 in 10% 0.002, 17.6% cells, mice mice mu/ mu mu

Journal of Cell Science C2cls loecneitniiso g eeqatfe rm6 ue-hN C2clsad5 oto el,rsetvl.*** respectively. cells, muted-s control Representa in 57 (H) increased and were experiments. cells CgA PC12 independent of muted-shRNA three intensities 63 mean least fluorescence from Average are at quantified (I) I were of compared cells. CgA and cells representative control of PC12 and are intensities muted-shRNA cells Fluorescence shown in of PC12 cells. increased immunoblots glands muted-shRNA PC12 drastically The in adrenal was CgA panel). in CgA of (lower Cellular levels images panel). immunoblotting SgII immunofluorescence (upper by in (CON) shown change cells as control significant to cells no relative was protein muted there of that reduction showed analyses immunoblotting of Quantification shown. are SgII oprdt oto ie( mice control to of compared glands of adrenal glands in peptide adrenal CgA-derived in in CgA increase full-length an in was increase an was there *** that showed analyses immunoblotting eeso g narnlgad in glands adrenal morphological in the CgA of of levels assembly mechanism an of molecular To as 2001). defects al., acting the et LDCVs, (Kim biogenesis investigate of granule secretory constituent drive to major factor a in is LDCVs cells CgA in PC12 CgA and of glands amount adrenal the increases deficiency Muted ARTICLE RESEARCH i.2 nrae g eesin levels CgA Increased 2. Fig. nieyt ecue yteiefcec fCAcevg.In cleavage. CgA unaltered were of SgII, granin, inefficiency full- another in of the of levels by protein increase in the caused contrast, CgA the be increased with the to that agreement unlikely suggests in This CgA. is length which peptide We CgA-derived 2C,D), 2A,B). (Fig. intermediate (Fig. an mice of ( con) amount (WT, the wild-type quantified to in kDa) compared (80 CgA mice full-length of (1.54-fold) increase significant , P mu 3ka n hr a ieiea nraein increase an likewise was there and kDa) 23 , .0.(,)Rpeettv etr lt fCAdrvdppie( peptide CgA-derived of blots western Representative (C,D) 0.001. iecmae ocnrlmc Fg EF.W confirmed We 2E,F). (Fig. mice control to compared mice mu 6 s.e.m. DV,w esrdtesed-tt protein steady-state the measured we LDCVs, n 5 ) .. o infcn.()Imnbotn nlsso yae rmsal ue-hN C2cls(hN)soe an showed (shRNA) cells PC12 muted-shRNA stable from lysates of analyses Immunoblotting (G) significant. not N.S., 9). mu dea lnsadmtdkokoncells. muted-knockdown and glands adrenal mu ie efuda found We mice. mu mu mu ie( mice ieis mice mice n mu 5 , )cmae ocnrlmc ( mice control to compared 8) 3ka r hw.Qatfcto fimnbotn nlsssoe htthere that showed analyses immunoblotting of Quantification shown. are kDa) 23 eils(rie l,18a oz n oz,1986). clathrin-coated Tooze, and via Tooze 1985a; (ISGs) CgA al., of et granules export (Orci secretory or vesicles removal immature the facilitate from might protein muted the of absence 5–50% complete a almost an through found western of sedimentation CgA by We CgA gradient. equilibrium of OptiPrep impaired after distribution from the non-LDCV blotting mislocalized monitored result from We might or organelles. LDCVs CgA immature increased from export the muted-KD that in cells reasoned CgA 2H,I). PC12 (Fig. of increased (KD) significantly intensities was muted-knockdown cells the in PC12 Consistently, CgA 2G). of (Fig. increase the yemc.I otat h itiuino gIwssimilar was SgII of distribution the wild- in contrast, with In between compared chain, mice. heavy type clathrin the to correspond AB ersnaiewsenboso g r hw.Qatfcto fthe of Quantification shown. are CgA of blots western Representative (A,B) stettlnme fLCsin LDCVs of number total the As ora fCl cec 21)18 3517 doi:10.1242/jcs.161414 1365–1374 128, (2015) Science Cell of Journal mu mu dea lnsi h ihe rcin n ,which 4, and 3 fractions lighter the in glands adrenal n Tmc Fg A.Teerslssgetthat suggest results These 3A). (Fig. mice WT and mu n ie( mice 5 ) * 8). n P 5 , 8 eaiet idtp cn ie( mice (con) wild-type to relative 28) .5 EF ersnaiewsenbosof blots western Representative (E,F) 0.05. mu el i o hne we change, not did cells P , .0.Rslsi ,D E D, B, in Results 0.001. mu ie( mice ocontrol to n n 5 , 5 hRNA 1367 tive 8) 90% 28).

Journal of Cell Science agl eandi h gIpstv DV nbt h muted-KD the both in LDCVs SgII-positive the in retained largely EERHARTICLE RESEARCH 1368 hs etdswr nihdi rcin –0 hc represent which that 7–10, observed fractions We in LDCVs. enriched and in kDa were concentrated (60 peptides peptides mainly these CgA are processed al., kDa) the et 23 regard, Orci this 2010; In major al., the et 1985b). as (Koshimizu considered sites been conversion have prohormone organelles. LDCVs other to mature mislocalized and or Immature LDCVs to localized was mice DVCAa ela oa g a ersudb the by rescued be the in can organelles LDCV CgA increased total the protein. muted that as functional suggest of well in expression results protein cells as These muted the 4D). EGFP-transfected LDCV-CgA of (Fig. LDCV-CgA expression the cells the the EGFP–muted- these verified with and We the 4B,C). compared CgA in (Fig. total cells decreased both significantly expressing that transfected muted- were the in showed proportion stable LDCVs intensities individual and the in the CgA quantified cells the We and into CgA 4A). total (Fig. of EGFP–muted cells PC12 transfected in knockdown increased we 3C). was (Fig. CgA, cells proportion control the LDCV with that the compared showed cells and in PC12 2014) muted-KD intensity al., et CgA (Walter granules LDCVs the we individual as this, cytosolic defined with in are CgA Consistent that of 3B). intensities (Fig. the mice quantified WT the with results in compared our proportion and LDCV-CgA the fractions total that LDCV-enriched the showed these quantified in then CgA We of 3A). amount (Fig. LDCVs of distribution the ete netgtdwehrCAi ilclzdt te non- other to mislocalized is CgA whether investigated then We in increase the to leads protein muted of loss that confirm To in CgA of amount increased the whether tested then We mu el.W bevdta g was CgA that observed We cells. mu iewsincreased was mice mu fCAi DV.A g a h blt oageaet form to aggregate to ability or in the content vesicles CgA has increased increase CgA the secretory the cores, As to electron-dense constitutive LDCVs. lead in thus to and CgA ISGs, of sorted in trapped be is might lysosomes which 5), (Fig. CgA cells of control unaltered. to is localization compared subcellular TGN its when in that (Rab7) suggesting (SEC61B), cells endosomes organelles muted-KD late (ER) and other the not (EEA1) reticulum endosomes did to early endoplasmic We (STX6), CgA LDCVs. the and of lysosomes including mislocalization both apparent of LAMP1, marker observe with a colocalization partial is a which with cells, control and cells eesbepo IP orsod oasbolo h R htis that RRP the of Ca subpool with associated a closely to immediately corresponds the (IRP) pool and releasable vesicles, represents fusion-competent generally of release RRP population the the the and cells, (RRP) neuroendocrine pool releasable In readily frequency. the of size the affect yterdcdIPadRPsz Fg AD.Teeresults cells. chromaffin These in and secretion 6A–D). size vesicular (Fig. RRP of smaller reduction a size a to in leads RRP results protein muted and of lack IRP that indicate reduced the by in reduced drastically was Average response cells. chromaffin and control single from obtained to responses applied was patch-clamp cell el ieylast h nrae ieo lcrndnecrsas LDCVs. cores of electron-dense size of increased size the increased as the well to leads likely cells etse hte h iecag nLC in LDCV in change size the secretion whether vesicle releasable in tested readily reduction We smaller a a and to size leads pool protein muted of Loss ae oehr nteasneo ue rti,teproportion the protein, muted of absence the in together, Taken ora fCl cec 21)18 3517 doi:10.1242/jcs.161414 1365–1374 128, (2015) Science Cell of Journal n h g loecneo ahvscewsmeasured. was vesicle each *** of fluorescence CgA the and eeslce n10 in selected were el (1.00 cells yooi eils ssoni h etpnl nmuted- increased in significantly panel, was left cells the PC12 individual in knockdown of shown 2B). CgA as Fig. of vesicles, in intensity cytosolic (shown fluorescence group mean control The the (C) of that the 1.54-fold for is 1.54 of the factor for a 1 by of the and factor in group, peak a control LDCV by the multiplied for was percentage sedimentations group The control the CgA. to total normalized and CgA total of percentage a (1.65 eas h vrg muto oa g nthe in CgA total of amount average the because mean and (con) wild-type from in levels glands CgA Increased 3. Fig. oto ie(1.00 mice lysates control in increased was peak LDCV from the in of kDa) amount (80 the CgA three that from showed blots sedimentations western independent nine peak. of in LDCV Quantification enriched the (B) were represent kDa) which 23 7–10, CgA- and fractions lysates. kDa control (60 not in peptides did that derived which to lysates, difference same any the show in SgII of distribution in chain was heavy to CgA clathrin corresponding that the fractions Note from blots. absent the completely immunoblotting; on almost by shown CgA are for fractions assayed 11 top and the gradient from the collected of were fractions 5–50% Fourteen through Optiprep. sedimentation equilibrium was by supernatant separated nuclei-free the and homogenized P , 6 mu 6 .0.Rslsaemean are Results 0.001. 0.02, ...CAcneti DVpa sepesdas expressed is peak LDCV in content CgA s.e.m. ie(1.61 mice 6 2+ 0.01, n 5 hnes(ot ta. 99.Whole- 1999). al., et (Voets channels 89vsce)cmae otecontrol the to compared vesicles) 4879 n 6 5 6 6 .2.*** 0.02). 64vsce) niiulvesicles Individual vesicles). 4694 0pxl ein 1pixel (1 regions pixels 10 .4 oprdwt hs from those with compared 0.04) mu mu mu iesoe htsecretory that showed mice hoafnclsa shown as cells chromaffin P yae scmae othe to compared as lysates , 6 .0.Rslsare Results 0.001. mu mu s.e.m. iewere mice LDCVs. mu mu el would cells chromaffin mu A Adrenal (A) 5 mu 2nm) 62 group group

Journal of Cell Science EERHARTICLE RESEARCH oprdwt h hrceitc fLCsi dysbindin-1 in LDCVs of characteristics deficient the LDCVs affect with to known adrenal Compared proteins in other levels from CgA differently steady-state glands the affects protein Muted hne nhl-egtdrto HD,rs ie(T and (RT) significant time no rise (HHD), found duration We half-height electrodes. in releasing carbon-fiber each changes of kinetics by the recorded spike We kinetics. normal with 6E,F). (Fig. mice control in the from to vesicles nm compared (0–100 docked vesicles the the is docked in of in maturation membrane) LDCVs number plasma LDCV of the enlargement when measured the We vesicles by evident docked as impaired, of reduction the 3) hs eut ugs httesalrRPpo osnot well-known does these proteins. of pool fusion levels or RRP steady-state docking the smaller in the changes that from result suggest results These S3A). levels state the steady in the proteins determined synaptic we the protein, of muted of population loss the This protein. by muted kinetics. the release by affected and be LDCVs the fusion not of might that normal population normal suggests have a This that represent S2). might vesicles Fig. and docked control material between (supplementary spikes amperometric mice of (Q) size quantal ytxn1adVM2,snpohsn(Y)adMunc18 and (SYP) and synaptophysin controls a (SNAP25, VAMP2), between of proteins levels SNARE and steady-state including the syntaxin-1 proteins in synaptic changes of apparent panel find not the did in observed We adrenal was in band protein no expected, muted As the cells. of expression the confirmed analyses oepoewehrtedcigo uinmcieyi affected is machinery fusion or docking the whether explore To release vesicles docked the whether that investigated then We from result could release vesicle or size RRP in reduction The sdy mice, mu mu iehv ifrn rfl fLDCV of profile different a have mice mu mu ie(upeetr aeilFig. material (supplementary mice mu el eesgiiatyreduced significantly were cells dea hoafncls Indeed, cells. chromaffin adrenal dea lns etr blotting Western glands. adrenal mu dea glands. adrenal mu cells. mu alr fCAepr ih marLC auainand maturation LDCV Fig. material impair (supplementary release might vesicle export finally Consequently, and 2006). LDCV CgA docking al., of of size et enlarged (Kim failure an LDCVs is general, when immature In it vesicles represents the lacking. rather from is export an protein processing, CgA from of muted failure result impaired the not by or does caused likely CgA number cores of dense vesicle increase and The increased LDCVs 2001). of al., sizes be et enlarged could (Kim which the electron increased, for morphology was explanation examining content an CgA LDCV by the protein, in muted docking) of reduced defects of images and microscopy the size characterized (enlarged we Here, DISCUSSION iecmae ihterwl-yecnrl.In controls. wild-type their with compared mice reduced is number cargo vesicle in in Notably, roles biogenesis. different LDCV play for muted sorting This both and complex. dysbindin-1 BLOC-1 although same that the suggests S1) in reside Table muted and dysbindin-1 material (supplementary phenotypes nraei DVnme.Ti ute uprsorhypothesis in our CgA supports of further increase the This that in number. CgA LDCV in in increase increase the the in that changed contrast, the not suggests and In was normal LDCVs 7). was SgII of (Fig. but number of significant increased levels was not the level steady-state in was CgA reduction this a also although was SgII there that and supplementary decreased was in SgII shown and CgA as of reduction SgII the and confirmed in We CgA S1. of Table lead material might decrease cells the chromaffin HOPS-deficient to or AP-3- in number nteepr fCA u a oo iteefc nSgII. on effect little or no has but role CgA, important of an export plays the protein in Muted LDCVs. immature in CgA pe sdy ( b iebti omlin normal is but mice ora fCl cec 21)18 3517 doi:10.1242/jcs.161414 1365–1374 128, (2015) Science Cell of Journal AA- eiiny and deficiency) AP-3 3A mu rnfce el xrs ue protein. muted express cells EGFP–Muted- transfected that showing Immunoblotting (D) ** cells. chromaffin 0.75 group, cnrl 1.00 (control, 0.54 counted. 5 were bar: signals Scale green EGFP (red). with CgA for Cells immunostained and hours more 72 for than (green) protein, an fusion express EGFP–Muted to pEGFP-C2-Muted, with or transfected pEGFP-C2 were Muted- cells content. PC12 CgA shRNA the reduced cells PC12 LDCVs. full-length the in content the CgA the reduced in cells protein PC12 muted muted-knockdown of Expression 4. Fig. fEF–ue eue h oa g level CgA 1.00 total (control, the reduced EGFP–Muted of expression that showed intensities fluorescence dea hoafncls nteabsence the In cells. chromaffin adrenal 6 mu 0.07, mu el scue ytertninof retention the by caused is cells Muted 6 n ie h euto fLDCV of reduction The mice. bf m 6 6 5 0.02, .(,)Qatfcto fCgA of Quantification (B,C) m. 0.02, 0.12, 4cls)adLC-g level LDCV-CgA and cells.) 64 VS3 OSdeficiency) HOPS (VPS33a mu DAi h muted-shRNA the in cDNA mu P n , 5 n n iei o u othe to due not is mice 5 5 .1 *** 0.01, 43vsce; in vesicles;) 1443 ie(is1 ) This 2). 1, (Figs mice 22vsce;Muted vesicles; 2222 4cls ue group, Muted cells; 64 A xrsinof Expression (A) sdy P , 0.001. ie CgA mice, mu 1369

Journal of Cell Science EERHARTICLE RESEARCH oti ohCAadSI.Frhroe u eut hwdan showed 1370 results granules our the Furthermore, of SgII. number and large CgA cells, both a control SgII- contain although and in exist, vesicles colocalize some vesicles CgA-positive not were positive separate there did that 5C, SgII supporting granins Fig. and further in these CgA shown for that As machineries granules 1991). The sorting al., 1991). implicates et different al., populations (Watanabe different et exist in Watanabe there SgII 2005; that and al., CgA et of Grabner segregation 2003; al., (Duncan et constituents different have LDCV might different which there exist, that populations postulated been has It process. regulated LDCV of machinery pathology. sorting than its being the rather and into cells and insights shRNA model in molecular sorting-for-retention LDCVs the provide the support in findings retained marker mainly These endosome was S3B). early CgA the that and and (E), the LAMP1 cells, (A), marker shRNA SEC61B lysosome in marker the increased ER (D), were [the STX6 CgA organelles. marker markers, of other TGN organelle intensities the to different (C), the mislocalized and SgII that CgA marker Note with LDCV (F)]. the co-immunostaining EEA1 (B), by Rab7 detected marker was cells. body PC12 CgA multivesicular muted-shRNA cells. in PC12 CgA (shRNA) of shRNA localization Subcellular 5. Fig. ag otn noLCsi eycmlctdadhighly and complicated very a is LDCVs into sorting Cargo ersnaieiae fsbellrlclzto fCAi oto CN n muted- and (CON) control in CgA of localization subcellular of images Representative nraei g u omllvlo gI n distributional a and SgII, the in of SgII, level not but normal CgA, a in shift but CgA in increase opeiyo utpergltr.I LC1 oso dysbindin- of loss BLOC-1, 1( In regulators. multiple of complexity (supplementary normal. is normal population LDCV is SgII-positive kinetics of events the releasing in release not S2) the and Fig. but material docking why CgA, vesicle of and why sorting exist explains the in also involved This is SgII. protein muted the that 08.I te Rs uha eaooe n platelet-dense and melanosomes as such both LROs, in granules, other numbers In in LDCV 2008). unchanged reduced is number to LDCV contrast the but size, LDCV oto fvscenme n iecudb elcino the of reflection a be could size and number vesicle of Control sdy n oso ue ( muted of loss and ) ora fCl cec 21)18 3517 doi:10.1242/jcs.161414 1365–1374 128, (2015) Science Cell of Journal mu and mu sdy hoafnclsgvnta h secretion the that given cells chromaffin ieso eue eil numbers, vesicle reduced show mice mu ohla oa nagmn of enlargement an to lead both ) mu dea lns hsindicates This glands. adrenal sdy ie(hne al., et (Chen mice mu ie in mice,

Journal of Cell Science ftwo of ihn10n rmtepam ebaewr eie smrhlgclydce eilsadcutd hr a oe rqec fmorphologically of frequency lower a was There counted. and vesicles docked morphologically as defined were membrane in plasma LDCVs the docked from nm 100 within ** mu EERHARTICLE RESEARCH ih as eue ertasiso n hratrthe thereafter and neurotransmission reduced regulating cause al., vesicles et synaptic in hippocampal Yuan isoform might of 2014; ultrastructure pathway al., dysbindin-1C altered et The 2015). (Wang BLOC-1-independent that survival neuronal shown a and autophagy recently in al., are have et distinct functions (Talbot subunits has pathways We dysbindin-1 different BLOC-1 in addition, 2009). function In might of 2007). that al., isoforms partners suggests et This (Li interacting 2007). operative al., others different et and Li that complex 2011; Dell’Angelica, WAVE-2 and contrast, the the (Ghiani to By complex, belonging proteins of protein mechanisms. variety a dystrophin sorting to bind to common they known is dysbindin although some pathways, share has cargo-sorting LRO might and each that biosynthetic indicates unique evidence mounting 2013). Li, together, and in Taken Wei mechanisms 1998; al., shared et utilizes (Swank trafficking LROs endo-lysosomal of biogenesis the indicating in reduction great 102.38 a of showed size average (D) an RRP observed and we (C) cells, chromaffin IRP control the In of IRP. the values includes averaged The (C,D) pulses. depolarizing of trains to response in cells chromaffin in LDCVs docked and secretion depolarization-induced Reduced 6. Fig. oprdwt oto.TeIPsz fec igecrmfi elwsdtrie stemxmmcpctnersoss( seventeen responses the capacitance during maximum responses the capacitance as maximum determined the was as cell determined chromaffin was single size each RRP of the four size whereas from IRP depolarizations, cells The (19 control. control with of compared increase capacitance Average (B) size. RRP the estimate from depolarizations P , hoafncls h R iewsrdcdby reduced was size IRP the cells, chromaffin .1.()Dsrbto fdsac ewe eil ebaeadpam ebaein membrane plasma and membrane vesicle between distance of Distribution (E) 0.01). mu / + el) * cells). mu 2 P (8.38 , 0t Vwt 0-sitrus nevlwr sdt siaeteIPsz,floe ysvnen50m eoaiain to depolarizations 500-ms seventeen by followed size, IRP the estimate to used were interval interpulse 100-ms a with mV 0 to 80 .5 eut r mean are Results 0.05. 6 0.65%, n 5 6 eilsfo 0clso two of cells 10 from vesicles 565 1 6 , s.e.m. 0 (60.04 40% 6 .7fF; 9.97 n mu/mu 5 2 * 22; mu ie el hnta ncnrl (11.05 controls in that than cells mice) P ehns nCAsrig g ncoti ielasto leads mice in knockout conserved CgA a sorting. share might CgA they in that mechanism suggesting islet S1), pancreatic Fig. in chromaffin material adrenal LDCV Our the the the both tissues. and in through different LDCV defects similar in shown systems biogenesis have DCV results nervous multiple of and regulation cardiovascular endocrine, consequences. in and defects mechanisms different vesicle have releasing might Thus, mice of 2009). profiles al., different et the (Gerrish schizophrenia for for support gene no susceptibility is there Currently, 2008). al., et (Morris in observed symptoms schizophrenia-like aen infcn soito ihshzprnaatog an although schizophrenia between with interaction association epistatic for significant except no However, 2014). have al., et Wang 2008; DTNBP1 al., et Feng 2008; hoafncells. chromaffin , 6 .5,adteRPsz a eue by reduced was size RRP the and 0.05), g a enipiae npaigmlil oe nthe in roles multiple playing in implicated been has CgA 92 FfrteIP( IRP the for fF 19.23 mu ora fCl cec 21)18 3517 doi:10.1242/jcs.161414 1365–1374 128, (2015) Science Cell of Journal +mc,bak and black) mice, /+ and mu ie( mice BLOC1S3 A ceai hwn h riso ih 50-ms eight of trains the showing schematic A (A) mu/mu n 5 9 n 354.84 and 19) ee noigohrBO- subunits BLOC-1 other encoding genes , n oto ie( mice control and ) mu 2 el rmfour from cells (22 DTNBP1 6 0.91%, , 0-spro.Nt htteRRP the that Note period. 500-ms 6 , 51 F( fF 35.19 D 0 (209.92 40% n m uigteeight the during Cm) and 5 5 eilsfo 2cells 12 from vesicles 054 2 mu a sdy cls(supplementary -cells MUTED +.()Vsce located Vesicles (F) /+). mu/mu n ie(hne al., et (Chen mice 5 9 o h R.In RRP. the for 19) 6 51 fF; 15.13 ie grey) mice, MUTED sdy ih exist might mu and , cells n 10-ms 1371 5 sa as mu 22;

Journal of Cell Science omte nEhc nteCr n s fLbrtr nml.The Animals. the Laboratory of ( from Use heterozygous and (+/+), type Care permission wild the University of Peking genotypes in under the Ethics and on Committee performed of Committee Use Institute and Care the were Animal of Institutional facility All experiments animal Sciences. of the Academy animal in Chinese Biology, bred Developmental are and (Buffalo,Genetics and Institute USA) Cancer Park York, Roswell New Dr at to laboratory transferred from Swank’s were obtained T. USA), originally Richard Maine, were Harbor, They (Bar 2004). Laboratory al., Jackson et the (Li al. et Li summarized ( sandy background, EERHARTICLE RESEARCH abt gis i-agdfso rti orsodn otemouse 1372 the white to Zealand corresponding New protein in fusion His-tagged generated a was against antibody rabbits muted polyclonal The Antibodies controls. background as used were mice heterozygous and Muted mu ( their pearl and mutants mouse including Other 2002). controls al., et (Zhang previously described The Mice METHODS AND MATERIALS Whether cells. chromaffin the reduced is from impairs secretion and CgA CgA frequency in our increase release In an 2005). causes LDCV al., protein muted et of (Mahapatra well loss animals as case, these cells, chromaffin in in hypertension LDCVs as of number and size decreased ikt yetnino te DVpeoye nti mutant this in phenotypes investigation. LDCV detailed other further or requires hypertension to link iewr dniidb C ehdbsdo h uaini the in mutation the on based method PCR a by identified were mice ) mu ee(hn ta. 02.Aemthdwl-yeCM/emice CHMU/Le wild-type Age-matched 2002). al., et (Zhang gene uatmc n oto HUL ie(idtp,W)wr as were WT) (wild-type, mice CHMU/Le control and mice mutant sdy nteDA2 DA akrudwr are were background (DBA) DBA/2J the in ) pe nvivo in n uf( buff and ) in mu ie rwehrteei a is there whether or mice, bf mu nteC7L6 (B6) C57BL/6J the in ) + n ooyos( homozygous and /+) mu/ #c208,GP(s-96 n otplcoa g (#sc-1488) CgA TX). polyclonal (Dallas, Biotechnology goat Cruz Santa CA). from and purchased Diego, SNAP25 were (#sc-9996) antibodies (San (#sc-12736), 1 GFP Biosciences syntaxin (#sc-20038), BD (#sc-17750), SYP LAMP1 from monoclonal monoclonal Mouse were rat the antibodies and monoclonal (#553792) (#610337) mouse Munc-18 The rabbit MO). (#610457), Louis, EEA1 The (St. Science Sigma-Aldrich MA). Life from monoclonal purchased Meridian (Danvers, mouse from The was TN). Technology SEC61B antibodies (Memphis, (#K55101R) (#4796) Signaling antibody and SgII chain rabbit Cell polyclonal heavy The from clathrin rabbit (#ab3347) UK). and were (Cambridge, the (#9367) VAMP2 Abcam Rab7 (#ab12370), from polyclonal obtained (#ab45179), 6 were (#ab78276) Syntaxin CgA mouse against The polyclonal NM_139063). antibodies RefSeq (GenBank monoclonal protein muted full-length dea lnsfo Tand WT from glands Adrenal assay gradient OptiPrep ECL NIH by by to quantified normalization were visualized after intensities J and bands Image Protein antibodies IL). specific Rockford, (Pierce, with immunoblotted membranes were protease The membranes. Millipore) onto a transferred (PVDF, and difluoride polyvinylidene gels and SDS-PAGE 12% on X-100, separated were homogenates Triton 1% 13,000 NaCl, g at centrifuged were extracts mM The (Sigma-Aldrich). 150 cocktail inhibitor 7.4, pH and Tris-HCl control from glands Adrenal Immunoblotting PS a eeae ycnrfgto t1000 at supernatant centrifugation a post-nuclear plus by 7.4) A generated pH was EDTA, (Sigma-Aldrich). mM (PNS) cocktail 1 Tris-HCl, inhibitor mM protein 20 sucrose, mM (250 buffer o 5mnt eoence n eldbi,eulaonsof amounts equal debris, cell and nuclei remove to min 15 for ora fCl cec 21)18 3517 doi:10.1242/jcs.161414 1365–1374 128, (2015) Science Cell of Journal in ftepoenlvl fCAadSI nadrenal in SgII and CgA of of glands levels protein the of uniaieaaye hwdadces in ( decrease CgA a showed littermates. analyses (B6) Quantitative C57BL/6J control their and ( ( CgA in a decrease showed analyses Quantitative (B6) littermates. C57BL/6J control their and deficiency) AP-3 n gIi h dea lnsof glands CgA adrenal of the levels in protein SgII the and of blots Western (B) significant not was the ( this in although reduction SgII, a of also amount was there that and eraei h muto g ( CgA of amount a the was in there decrease that showed littermates. analyses DBA/2J Quantitative control their the and in deficiency) SgII and of CgA glands of adrenal levels protein the the in of SgII mice. and HPS CgA of glands of adrenal content The 7. Fig. n n 5 5 bf 2 * 12, ,NS,ntsgiiat in significant) not N.S., 8, mu b ie eut r mean are Results mice. n -actin. 5 mu iewr ooeie n300 in homogenized were mice P ,* 4, bf , iewr ooeie n5 mM 50 in homogenized were mice .5 in 0.05) P ie(P3aHP deficiency) HOPS (VPS33a mice , .5 n gI( SgII and 0.05) b atnatbd #54)was (#A5441) antibody -actin sdy n 5 pe 2 ** 12, ie(ybni BLOC-1 (dysbindin mice ie D etr blots Western (D) mice. g o 0mn h PNS The min. 10 for P , 6 sdy .1 n SgII and 0.01) A etr blots Western (A) s.e.m. n n 5 pe 5 mice. ,** 6, ,* 8, ie( mice P P , , m 0.01) 0.05) b lHB 3A

Journal of Cell Science utrda 37 at cultured elclueadtransfections and culture Cell rotor SW41 an in hours 15–16 4 for at rpm USA) 30,000 USA). (Biocomp, at (Beckman, Master (Axis-Shield, spun Gradient a gradient were using Optiprep Gradients buffer linear HB 5–50% in formed linear Norway) a onto layered was ARTICLE RESEARCH 08.Bify dea lnsrmvdfo homozygous from al., removed heterozygous et glands (Chen adrenal described previously Briefly, as 2008). out carried was microscopy Electron microscopy Electron 4 at hours 16 for incubated then min PBS, 30 in for blocked BSA PBS, in temperature, 1% X-100 room Triton at with 0.4% min with min 20 15 for for hours. paraformaldehyde permeabilized 4% 24 with for fixed coverslips were glass Cells poly-D-lysine-coated on plated were cells PC12 imaging confocal Immunofluorescence 37 at cultured were cells PC12 ufr(nm:14NC,56Kl . EE,36NaHCO 3.6 HEPES, 5.0 KCl, 5.6 NaCl, 154 mM: (in buffer 6- from heterozygous dissected 8-week-old were to glands Adrenal modifications. (Life minimal with 2003) transfection. system after hours transfection 72 instruction. and immunostaining Neon coverslips for manufacturer’s glass processed poly-D-lysine-coated the the then onto seeded to were using cells according Transfected by CA) Carlsbad, transfected Technologies, the were using pEGFP-C2 into Cells in-frame cloned clone and cell PCR transfected the 5 by hours, selected 24 was vector After the expressing instructions. manufacturer’s the mgswr curduiga100 a Confocal using BSA. acquired 1% were containing images PBS in donkey Invitrogen) Alexa-Fluor-488-conjugated (1:2000; IgG or anti-rabbit Invitrogen) (1:2000; IgG Alexa-Fluor-594-conjugated goat Alexa-Fluor-488-conjugated anti-rat Alexa-Fluor-488-conjugated Invitrogen), Invitrogen), (1:2000; Invitrogen), IgG (1:2000; anti-goat donkey IgG (1:2000; subsequently anti-rabbit donkey were donkey Alexa-Fluor-594-conjugated IgG Cells a with BSA. hour anti-mouse 1 1% for containing incubated and PBS washed in antibodies primary eul ne ugclmcocp.Temdla eeicbtdin incubated were O medullae (95% The equilibrated microscope. adrenal ml from surgical 0.2 removed was a cortex under adrenal and medulla tissue Fat 7.4). pH glucose, ok’ ufr h aanwsfo im-lrc)a 37 at Sigma-Aldrich) from was papain the buffer; Locke’s olce rmtetpo h rdetadsbetdt acetone to subjected and gradient by analyzed the was immunoblotting. 3–13) of and (fractions SDS-PAGE protein top precipitated The the precipitation. from collected C2clsuigLipofectamine using cells for PC12 sequence target AATTCTCCGAACGTGTCACGT-3 The vector. AAGGATCTTGGAGAAATTCAT-3 (Ambion) Retro 5.1-H1 (shRNA) RNAs rat fetal hairpin against 5% Short MD). and Gaithersburg, serum (Gibco-BRL, horse serum 10% bovine with supplemented (DMEM) medium Eagle’s ihgnl hkn.Teezm ouinwsdsaddadrpae with replaced and discarded was 500 solution enzyme The shaking. gentle with ltrleyei . hsht ufr(H74 vrih n were and overnight 7.4) (pH buffer phosphate M 0.1 in glutaraldehyde h eceeprmn,the experiment, rescue the bandfo h niecl,tenceswssbrce n mg was For image and Nikon. software. subtracted J was by Image nucleus NIH was the provided with signal cell, analyzed fluorescence entire software the the from intensity, 3.2 obtained fluorescence obtained the AR were of NIS-Elements Images quantification Japan). the Ti-C2, using (ECLIPSE microscope confocal yteie n noprtdit the Aust into incorporated (Ambion, and synthesized finder target siRNA 0 Um eiilnad100 and penicillin IU/ml 100 ie n500 in times hog 10- a through yiecae ls-otmddse oalwteclst etefr3 min 30 for settle to 37 cells the at allow to dishes glass-bottomed lysine-coated hoafnclswr sltda rvosydsrbd(øesne al., et (Sørensen described previously as isolated were cells Chromaffin m ˚ eoesplmnigwt lcluemdu.Teclswere cells The medium. culture ml 2 with supplementing before C utr eim(MMsplmne ih1%ftlcl serum, calf fetal 10% with supplemented (DMEM medium culture l Muted m ˚ m Cin5%CO mu iet i.50 tip. pipette l utr eim h eulewr rtrtdgently triturated were medullae the medium, culture l GnakRfe M01037 eedsge ythe by designed were NM_001107347) RefSeq (GenBank +ltemts( ek l)wr ie ih3% with fixed were old) weeks (8 littermates /+ ˚ .Fute qa rcin (900 fractions equal Fourteen C. 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Differences groups. statistically two of means compare to oeta f8 V h nrclua ouincnand(nm) Cs- mM): (in holding contained current solution direct intracellular a The about mV. peak- 80 applied mV of was 20 and potential Hz, amplifier stimulus 800 EPC-9 sinusoid A Germany). an to-peak Lambrecht, using (HEKA, software performed Pulse were recordings cells Whole-cell chromaffin of measurements Capacitance upeetr aeilaalbeoln at http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.161414/-/DC1 online available material Supplementary material Supplementary KJZD-EW-L08]. number [grant Sciences of Academy the Chinese and 2014CB942803]; 2013CB530605, Science numbers [grant of China Ministry of Science Technology the 91332116]; and Natural 31230046; National numbers the [grant from China grants of Foundation by supported partially was work This Funding recordings. Z.H. amperometric and W.D. and X.C., electrophysiological immunostaining, and L.W., the the W.L. assays. performed performed data. microscopy J.M. the electron and analyzed Y.F. and and Z.H., immunoblotting assays manuscript. the the designed wrote Z.H. Z.H. and Z.Z. L.C, W.L., contributions Author interests. financial or competing no declare authors The interests Competing taking and We samples manuscript. microscopy this electron HPS making of the images. in proofreading of microscopy help his her providing electron for for generous Sun and his Lei study for Ms. this thank Swank T. for used Richard Dr. mice to mutant debt in are We Acknowledgements mean as shown are text the in values All 1996). USA) Misler, Statistics and (WaveMetrix, (Zhou software program Igor macro using custom-made out a carried with was only analysis (Q), size Data the quantal and including were (RT) that spikes, time events rise amperometric the of (HHD), properties duration half-height kinetic For Germany). the amplifier Lambrecht/Pfalz, of EPC9/2 Elektronik, an analysis using (HEKA by software 2008) Pulse al., and et recordings (Chen whole-cell previously performed described and as slices medulla adrenal prepared We slice adrenal in Amperometry MgCl 8; NaCl, 145; glutamate, xeiet eepromda omtemperature. room at performed were Experiments aa)a 0k.Dgtliae eetknfo admyselected genotypes. randomly both from for taken animals JEOL, were different 1400; two images (JEM of Digital microscope cells kV. electron 80 an at on Japan) examined Ultra-thin were PA). Chester, sections West (SPI-Chem, ( Spurr sections in embedded were and EE,1 p .) h xrclua ouincnand(nm)NaCl, mM) (in MgCl contained 2.8; solution KCl, extracellular 140; The 7.2). (pH 10 HEPES, t4 at %prfradhd n .%guaadhd n01Mphosphate M OsO 1% 0.1 with post-fixed in were and glutaraldehyde overnight 7.4) 2.5% (pH buffer and paraformaldehyde 2% Epon812. in embedded were and ethanol ( of sections Ultra-thin series graded a in dehydrated otie ih1 OsO 1% with postfixed ydrc esrmn.Teosre a ln otegenotypes. the to blind was observer determined The were measurement. cores direct electron-dense by and diameter vesicles a the of in with included Diameters Vesicles were analysis. area. core electron-dense cytoplasmic an cell and membrane chromaffin per round the LDCVs different identifiable granules by morphologically two secretory divided of section number the of the of as cells density calculated Numerical chromaffin was images genotypes. digital selected both 100 for randomly least animals At from electron J. 20 taken Image Tecnai NIH were FEI with a analyzed on examined and were microscope sections The citrate. lead and h sesfo age-matched from islets The ˚ .Tsu ape eete eyrtdi rddsre facetone of series graded a in dehydrated then were samples Tissue C. ora fCl cec 21)18 3517 doi:10.1242/jcs.161414 1365–1374 128, (2015) Science Cell of Journal , 0n)wr olce n tie ihla irt.The citrate. lead with stained and collected were nm) 70 . ..o h os eeue Zo n ilr 1996). Misler, and (Zhou used were noise the of s.d. 5 , 2 0n)wr olce n tie ihuay acetate uranyl with stained and collected were nm) 60 ;CaCl 1; , 4 o or t4 at hours 2 for 2 mu 2 ;HPS 0 n lcs,2(H7.4). (pH 2 glucose, and 10; HEPES, 2; , ;M-T,2 Na 2; Mg-ATP, 1; , +and /+ 6 mu/mu ...Student’s s.e.m. ˚ .Tsu ape eethen were samples Tissue C. P aemc eefxdin fixed were mice male , .5wr considered were 0.05 2 GP .;adCs- and 0.3; -GTP, t ts a used was -test 4 o hours 2 for 1373 a -

Journal of Cell Science ra,P n ate D. Castle, and P. Arvan, J. A. Merz, and G. C. Angers, References ARTICLE RESEARCH 1374 C. E. Dell’Angelica, and and C. A. M. C. O’Donovan, Ghiani, J., M. Owen, V., Moskvina, H., Williams, A., Gerrish, Falco Apps, G., Bodammer, I., Matskevich, K., U. Wiegand, J., Greaves, R., R. Duncan, ae,M . ro,K,Stuuahn .adLvn,T P. T. Levine, and J. Satkurunathan, K., Bryon, J., M. Hayes, wn,B,Mria .A,Bnfcn,J . vdrky,E . Lamoreux, V., E. Sviderskaya, S., J. Bonifacino, A., J. Martina, B., Gwynn, P. A. Fox, and L. A. Cahill, A., P. Lysakowski, A. D., S. Fox, Price, P., and C. Grabner, A. Lysakowski, D., S. Price, P., C. Grabner, Zhen, Y., Guo, J., C. Hao, L., X. Guo, H., Wang, X., He, Y., Z. Zhou, Q., Y. Feng, Bonifacino, A., W. Gahl, M., Huizing, K., Moriyama, B., Gwynn, L., S. Ciciotte, Huang, N., Guo, Y., Z. Zhou, X., He, L., X. Guo, J., C. Hao, Q., Y. H. Feng, W., Z. X. Sheng, Chen, and H. Qiao, B., Y. Zhu, H., J. Brodsky, Tian, L., L., Lu, T. 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