oo complex motor Ato o orsodne([email protected]) correspondence for *Author n eeomna ilg,CieeAaeyo cecs ejn 100101, Beijing Sciences, of Academy China. Chinese Biology, Developmental and eLi Ke p150 dynactin with interacts HPS6 ARTICLE RESEARCH ß 4574 2014 August 22 Accepted 2013; August 30 Received al., et Watson other 2009; many 1 al., for proteins et adaptor 2005), Wassmer Chao, 1999; 2007; and al., et Yano al., Tai 2009; 2005; 2007; al., et al., et et Johansson Hong Traer 2001; 1997; al., al., et et Hoogenraad have (Engelender have trafficking cargoes characterized intracellular in been membranous tether functions their its that and proteins identified to been several complex 2009) Although al., cargoes. dynein–dynactin et to Moore the it 2000; links Schroer, also and King (Culver- but 2006; tracks not al., microtubule that et along complex Hanlon processivity protein its multisubunit cell enhances a require only the dynactin, dynein of of from Functions activity 2011). materials the (Allan, center of the primarily transport to is periphery and retrograde microtubules for of ends a responsible of minus range dynein, the wide towards a Cytoplasmic organelle of cargoes movement for the function. drives essential motor, and microtubule-based is which homeostasis trafficking, crucial biogenesis, plays membrane proteins in motor by roles driven transport intracellular The INTRODUCTION p150 HPS6, WORDS: KEY the with interacts directly organelles. HPS6 lysosome-related that of p150 that biogenesis demonstrate dynactin complex protein the we originally BLOC-2 in Here, the has of involved subunit (HPS6) is a protein as identified 6 been syndrome Hermansky-Pudlak ABSTRACT P6i eltd ugsigta eiula positioning for so- perinuclear required a uncovered is have that results complex Our activity. when motor and and/or suggesting maturation dynein–dynactin impaired lysosome endosomes the also depleted, by late are mediated is between lysosomes fusion and HPS6 as bodies degradative well multivesicular and protein as acidification lysosome-mediated capacity, in lysosomal delay Moreover, causes degradation. of also of movement knockdown centripetal but blocks partially perinuclear lysosomes (siRNA)-mediated only not the cells RNA HeLa to in periphery HPS6 interfering the cell mediate Small the to motor from region. retrograde lysosomes the of for adaptor transport cargo as acts and a-nnw pcfcrl o P6i h pta itiuino the of distribution spatial the in compartment. HPS6 lysosomal for role specific far-unknown rfikn n auaino lysosomes of maturation and trafficking tt e aoaoyo oeua eeomna ilg,Isiueo Genetics of Institute Biology, Developmental Molecular of Laboratory Key State 04 ulse yTeCmayo ilgssLd|Junlo elSine(04 2,47–58doi:10.1242/jcs.141978 4574–4588 127, (2014) Science Cell of Journal | Ltd Biologists of Company The by Published 2014. 2 1,2 nvriyo hns cdm fSine,Biig103,China. 100039, Beijing Sciences, of Academy Chinese of University i Yang Lin , Glued uui ftedni–yatnmtrcomplex motor dynein–dynactin the of subunit 1 hn Zhang Cheng , Glued CN,lssm,dynein–dynactin lysosome, DCTN1, , 1 agNiu Yang , 1,2 e Li Wei , 1 n i-i Liu Jia-Jia and flssmsrl pnmlclrmtr,adbt h ci and actin the both and cholesterol motors, distribution al., molecular upon steady-state et rely and and lysosomes Settembre trafficking of 2009; intracellular clearance The Klumperman, 2013). and pathogen signaling, and (Saftig receptor-mediated cellular homeostasis presentation, many as for such antigen essential processes, are of physiological and degradation and the pathways, phagocytic in endocytic, autophagic and from role of received acids are central that degradation a macromolecules nucleic play for lipids, They required polysaccharides. proteins, enzymes including hydrolytic macromolecules, discovered. be contain to remain that center the to periphery transported cell retrogradely the are from that cargoes vesicular and organelles yendnci oo ope.W rsn vdneta HPS6 p150 that dynactin to evidence binds present We complex. BLOC-2 motor of dynein–dynactin functions 2013; molecular al., biological exact et the elusive. – Peter – most remain John 2007) 2010) subunits identified al., 2012; al., been et al., have et Kloer et BLOC-3 Setty (Gerondopoulos and 2003; recently BLOC-1 of al., of al., biogenesis et et functions the Pietro Di Martina for 2003; Zhang al., 2006; important 2013; et established are (Chiang Li, been organelles complexes has and lysosome-related it BLOC Wei Although 2003). 2003; the al., al., 2004; that et HPS9 al., Zhang et 2002; et Li and al., Pietro 2004; et (Di HPS8 al., BLOC-3 et constitute HPS7, BLOC-2; Gautam HPS4 constitute and HPS6 these, HPS1 and and HPS5 Of HPS3, BLOC-1; BLOC-3. constitute and known (BLOC)-1, are complex that BLOC-2 organelles complexes lysosome-related of protein of biogenesis forms three as eight of other subunits the in encode mutated HPS genes (AP-3), known 3 (also complex gene (AP) HPS2 nine the these for in Except occurs as 2013). of that Li, and and loss biogenesis (Wei bleeding, partial forms prolonged by the and characterized pigmentation is in human of that a disorder Defects (HPS), recessive syndrome autosomal 2007). Hermansky–Pudlak others cause many al., organelles and bodies et lamellar platelet- (Raposo granules, melanosomes, include lytic They al., granules, 2007). et dense al., (Marks et functions Raposo cell-type-specific 2013; store for but proteins lysosomes, secret with and features many share that the compartments 2011), unexplored. al., largely of remains mTOR clustering et order cells steady-state in through (Korolchuk lysosomes perinuclear in nutrients of flux relevance to positioning autophagic physiological responses and intracellular that cellular signaling reported been coordinate their has Munro, to it and change Although Rosa-Ferreira 1998). 2004; lysosomes al., Jordens al., et 1998; et Santama al., Matsushita et 2011; Cantalupo Harada 2001; 2001; 1997; al., al., et et al., Cordonnier 2001; et al., et (Burkhardt cytoskeletons microtubule Glued yooe r aaoi raelso uaytccells eukaryotic of organelles catabolic are Lysosomes nti td,w on htHS sacroaatrfrthe for adaptor cargo a is HPS6 that found we study, this In tissue-specific of class a are organelles Lysosome-related AP3B1 hc noe the encodes which – ) omdaeretrograde mediate to 1, * Glued as nw sDT1,adta this that and DCTN1), as known (also b sbnto h dpo protein adaptor the of -subunit

Journal of Cell Science -emnso p150 of C-terminus Fg A,idctn htteitrcinbtenHS and HPS6 between interaction the that p150 indicating 1A), (Fig. hwdta h -emnlp150 C-terminal the that showed LC2cmlx–btas p150 also but and the lysates of – subunits two cell other complex the HeLa – HPS5 with BLOC-2 and HPS3 co-IP only not performed detected we interaction, etasetyc-rnfce E23clswt plasmids p150 with the cells full-length verify HEK293 epitope-tagged To co-transfected HPS6. encoding p150 transiently of an and encode we HPS6 66–197) to found between was (aa interaction one clones, fragment positive 20 N-terminal Of 2009). al., et utrdclsfie oc-muorcptt ihp150 with co-immunoprecipitate in expressed to were failed that 1–700) cells and N-terminal 1–500 cultured not the 1–400, were of (aa all HPS6 surprisingly, fragments of cells, mammalian fragments in 1G). N-terminal expressed (Fig. extreme assay two-hybrid the yeast Although the in verified was 1138–1281) noyi age.Ti oee a ersudby rescued be truncation HPS6 can by p150 – bind not to unable however but are of that mutants HPS6 – degradation full-length lysosomal This of impaired overexpressing caused also cargoes. positioning to only but endocytic lysosomes not periphery perinuclear LAMP2-positive cells cell and HeLa the the LAMP1- in of HPS6 for mislocalization of required Depletion lysosomes. is interaction ARTICLE RESEARCH Fg H,sgetn httergo daett h xrm N- extreme p150 the with to interaction adjacent its region inhibits the terminus that suggesting 1H), (Fig. p150 co-expressed that of cells co-IP from and extracts assays cell two-hybrid yeast by HPS6 on site(s) interaction dynactin human of brain C-terminus fetal human the a using of we by p150 screen motor, library dynein–dynactin two-hybrid the cDNA yeast for a adaptors cargo performed of search In p150 dynactin the with interacts directly HPS6 RESULTS xrm -emns eas tepe omptep150 the map to attempted also We C-terminus. extreme xeiet nclue amla el ihasre of co-IP series a and with 1D) cells (Fig. mammalian assays cultured p150 two-hybrid in experiments yeast performed P6(a6–0)adteeteeCtriu fp150 of C-terminus of extreme region the and N-terminal 66–200) the (aa between HPS6 Interaction fragments. HPS6 rmclsta oepesdHS n p150 and HPS6 co-expressed that cells from noyi age olssms o yooemtrto and maturation lysosome for lysosomes, of functioning. delivery to the cargoes for crucial that endocytic lysosomal is We, and lysosomes lysosomes, retrograde of to cells. positioning motor perinuclear facilitates retrograde HPS6-depleted the HSP6 linking in by that trafficking impaired and propose LEs/MVBs) and/or also therefore, as endosomes were to late referred lysosomes between (hereafter fusion bodies as multivesicular well as acidification niaeta h P6bnigst np150 data on these site Together, HPS6-binding 1E,F). the (Fig. that HPS6 indicate with strongly interacts rti odn.Itiunl,aogtetucto mutants truncation was 201–805) p150 the bind (aa to fragment able among truncated N-terminally an Intriguingly, expressed, folding. protein ihimblzdatbde gis h lgtg P6and HPS6 tag. Flag the (co-IP) against co-immunoprecipitation p150 antibodies immobilized performed with and respectively, nemdaecan(eefe eerdt sDC nthe in DIC) as to 1B). referred (Fig. HPS6 (hereafter of immunoprecipitates chain intermediate ftedni–yatncomplex dynein–dynactin the of ompteHS neato ieo p150 on site interaction HPS6 the map To Glued Glued Glued Glued mn cdrsde a)7817,a at(Hong bait as 718–1278, (aa) residues acid amino , loocr nmmaincls ofrhrvrf the verify further To cells. mammalian in occurs also rget.I amla el,atog h extreme the although cells, mammalian In fragments. eirclyc-muorcpttdwt ahother each with co-immunoprecipitated reciprocally Glued Glued n nte -emnltucto fragment truncation N-terminal another and , a o xrse,c-Po elextracts cell of co-IP expressed, not was Glued Glued Glued rgeto a911–1281 aa of fragment utemr,lysosomal Furthermore, . Glued Glued nmmaincells, mammalian in Glued Glued Glued n h dynein the and hog aberrant through Glued Glued n ait of variety a and Glued eie nits in resides n HPS6, and eagain we , fragments subunit Glued Glued Glued (aa rvnb h yendnci oo complex. motor the dynein–dynactin that the establish by HPS6 data driven of our distribution Therefore, compete 2007). perinuclear cannot al., thus, et wild-type and, (Johansson endogenous, itself the by complex with dynactin the proba into 2B), (Fig. effect obvious h bec ftecrorcgiinst,teN-terminal the dynactin, site, in of cargo-recognition that its p150 the subunit reasoned of mediate we of Arp1 fragment 2004), regions absence the (Schroer, middle the and 1C) (Fig. DIC and respectively with N-terminal interaction its because Flag-p150 a 0–0)soe ekp150 weak showed 301–805) (aa yokltnadatvt ftedni–yatnmotor dynein–dynactin the of activity microtubule and intact cell an the both cytoskeleton to requires signals vesicles HPS6-associated HPS6 th of of indicating dispersal 2B), (Fig. caused periphery also 2007), dynamitin al., (Ec dynein– et p50 complex the disassembles 2A). protein Flag-tagged which (Fig. dynactin dynactin, periphery a of cell subunit of the (DCTN2) to overexpression signals nocodazole HPS6 Moreover, Indeed, of motor. which dispersal disassemb retrograde and cytoskeleton, in the resulted microtubule for treatment track the as disrupt serves to c motor nocodazole To mediated HPS6-associated dynein–dynactin transport of the S1). retrograde by distribution requires Fig. perinuclear structures membranous material the whether (supplementary perinuclear antibodies test to localized structures with primarily it cells vesicular that revealed HeLa HPS6 of against staining the Immunofluorescence of activity requires complex HPS6 dynein–dynactin of distribution perinuclear The GLA)wsosre Fg AB.Hwvr o only not However, 3A,B). (Fig. observed marker p150 most was endosome the early (GOLGA4) of the the two or and EEA1 little HPS6 3A,B), contrast, (Fig. between In 2006). LAMP2) colocalization (Eskelinen, proteins and lysosomal (LAMP1 abundant (also 1 proteins 2 membrane CD63 lysosome-associated and with colocalized marker extent HPS6 higher Intriguingly, a LE/MVB 3A,B). to by (Fig. the LAMP-3), cells HPS6 as with known microscopy. HeLa colocalized confocal in partially and distribution staining analyzed compartmental immunofluorescence first we subcellular HPS6, of its function(s) cellular the compartment investigate lysosomal To the with associates HPS6 complex. oehr h idn htHS neat ihp150 with interacts HPS6 that finding the together, st yohsz htHS evsa ag dpo omdaethe complex. mediate motor dynein–dynactin to the adaptor by cargo driven transport as retrograde serves HPS6 that hypothesize to us fC-P rmedsmst h G sdsutdby disrupted is TGN the p150 to a endosomes of SNX6- from that overexpression found CI-MPR we of the study, transport in previous retrograde protein dynein–dynactin-complex-driven our mediated, full-length In the complex. with motor compete to mutant negative ipra fHS.Hwvr vrxrsino t HPS6- its of Flag-p150 cytoplasmic 911–1281, overexpression (aa causing However, C-terminus HPS6. our thereby binding in of fragment distribution, found protein dispersal we same HPS6 this, the with of perturbed overexpression agreement that In study 2009). current al., et (Hong eas p150 Because Glued ora fCl cec 21)17 5448 doi:10.1242/jcs.141978 4574–4588 127, (2014) Science Cell of Journal u loLM1adLM2wr eetdon detected were LAMP2 and LAMP1 also but , Glued trans N htlcsteSX-idn C-terminus SNX6-binding the lacks that -N) Glued Glintok(G)rsdn rti p230 protein (TGN)-resident network -Golgi Glued id oHS hog t -emns and C-terminus, its through HPS6 to binds ln ol ev sadominant- a as serve could alone Glued eer ta. 96 Melkonian 1996; al., et heverri l eas tcno assemble cannot it because bly yo h irtbl network microtubule the of ly rnainmtn a 1–910, (aa mutant truncation mlx etetdclswith cells treated we omplex, Glued tteprncerdistribution perinuclear the at rti o eiua cargoes vesicular for protein eursrtord transport retrograde requires idn Fg H.Taken 1H). (Fig. binding Glued Glued C a no had -C) prompted 4575

Journal of Cell Science EERHARTICLE RESEARCH ebaefatoso eacls(i.3) Moreover, 3C). (Fig. 4576 membrane cells from from HeLa vesicles immunoisolated LAMP1-positive of of been immunoisolation fractions had that membrane vesicles HPS6-positive p150 with interacts directly HPS6 1. Fig. nu n on rtiswr nlzdb D-AEadimnbotn ihatbde gis p150 against antibodies with immunoblotting and SDS-PAGE by analyzed were proteins bound and Input oI ihimblzdFa nioy(et.Cnesl,HK9 el vrxrsigFa-agdHS n y-agdp150 Myc-tagged and HPS6 Flag-tagged overexpressing cells HEK293 Conversely, (left). antibody Flag immobilized with co-IP C igasdpcigtepiaysrcue fp150 of structures primary the depicting Diagrams (C) rnfre ihpBT n T11o GK75 n T11srea eaieo oiiecnrl,rsetvl.()C-Pfo E23clsoverexp cells HEK293 from Co-IP (E) respectively. controls, positive p150 or Flag-tagged negative various as and serve pTD1-1 HPS6 and Myc-tagged pGBKT7-53 or pTD1-1 the and pGBKT7 and with medium transformed high-stringency on cells yeast co-transformed idn ie aebe dniid DYattohbi sa hwn h neato fHS ihp150 with HPS6 of interaction the showing assay two-hybrid (D)Yeast identified. been have sites binding o oI ihimblzdFa nioy(ih) nu n on rtiswr nlzdb D-AEadimnbotn ihatbde gis Myc against antibodies with immunoblotting and SDS-PAGE by analyzed were p150 proteins endogenous bound of and Co-IP Input (right). (B) antibody Flag immobilized with co-IP for a eemndwt I mgJ aawr rmtreidpnetc-Peprmns G es w-yrdasysoigteitrcinbtenHPS between interaction the showing assay two-hybrid Yeast (G) experiments. co-IP independent three from were p150 and Data mutants ImageJ. deletion NIH with determined was E Glued Glued -emns H oI rmHK9 el vrxrsigMctge p150 Myc-tagged overexpressing cells HEK293 from Co-IP (H) C-terminus. n I rmHL ellstswt nioisaantHS.Nra g n nioist N1srea controls. as serve SNX1 to antibodies and IgG Normal HPS6. against antibodies with lysates cell HeLa from DIC and Glued . Glued A E23clsoeepesn lgtge p150 Flag-tagged overexpressing cells HEK293 (A) eeinmtns F uniiaino rti-rti neato ewe P6adp150 and HPS6 between interaction protein-protein of Quantification (F) mutants. deletion Glued n P6 oan n idn ie np150 in sites binding and Domains HPS6. and a Glcoiaeclrmti sa speiul ecie Hn ta. 09.Yatclsco- cells Yeast 2009). al., et (Hong described previously as assay colorimetric -Galactosidase rcin hwdta o nyLM2btas P6and HPS6 also but LAMP2 only not that p150 showed fractions ofrhrdtriewehrHS s ned soitdwith associated indeed, is, HPS6 whether determine further To Glued ora fCl cec 21)17 5448 doi:10.1242/jcs.141978 4574–4588 127, (2014) Science Cell of Journal soit ihLM1pstv yooe Fg 3D). (Fig. lysosomes LAMP1-positive with associate Glued Glued Glued Glued n y-agdHS,SX rSX eelsdfor lysed were SNX1 or SNX6 HPS6, Myc-tagged and Glued -emns neato a eetdb rwhof growth by detected was Interaction C-terminus. I,HS,HS,HS,SX n SNX6. and SNX1 HPS5, HPS3, HPS6, DIC, , r niae.FrHS okondmisand domains known no HPS6 For indicated. are n aiu lgtge P6dlto mutants. deletion HPS6 Flag-tagged various and Glued P5o N6wr lysed were SNX6 or HPS5 , Glued rget in fragments n Flag. and ressing 6

Journal of Cell Science ojgtdbas(i.3) oehrteedt niaeta HPS6 lysosomes. that with indicate data associates these Together GM130- 3E). (Fig. with beads associated conjugated found were structures Golgi-cisternae-like EERHARTICLE RESEARCH itiue nclsta a endpee fHS Fg 4A,B). (Fig. evenly HPS6 of were depleted LAMP2 been had or that LAMP1 cells RNA with in distributed showed interfering labeled microscopy and lysosomes (supplementary small Immunofluorescence that transport S1E,F). (RNAi) by Fig. lysosomal interference material HPS6 in the RNA in depleted HPS6 (siRNA)-mediated role of we a role(s) positioning, plays To the lysosomes. HPS6 of investigate distribution associates that subcellular HPS6 and reasoned transport that retrograde we and lysosomes, distribution with perinuclear motor for dynein–dynactin the complex requires HPS6 that established Having lysosomes of and positioning motility perinuclear microtubule minus-end-directed mediates HPS6 the IgG- against was with antibodies immunoisolation association when with contrast, in performed In 3E). detected (Fig. beads were whereas conjugated beads, structures transmission HPS6-conjugated vesicular with associated by no be structures to beads lysosomal found were lamellar magnetic Electron-dense microscopy. on electron vesicles immunoisolated of HPS6-associated analysis ultrastructural performed we lysosomes, 20 or intact DMSO an with activity. requires treated dynein-dynactin HPS6 and of network distribution microtubule perinuclear The 2. Fig. rnfce ihFa-agdp0 p150 p50, Flag-tagged with and transfected HPS6 against antibodies with a9118)fr2 n muotie ihatbde gis HPS6 against 10 antibodies bar: with Scale immunostained Flag. and and h 24 for (aa911–1281) m m. m ooaoefr1h ie n immunostained and fixed h, 1 for nocodazole M a tbln B eaclswr transiently were cells HeLa (B) -tubulin. Glued N(a–1)o p150 or (aa1–910) -N cis Glimre GM130, marker -Golgi A eaclswere cells HeLa (A) Glued -C hc abra nenldlto a 8–8)mtn fHPS6 of mutant 187–189) p150 (aa with interact deletion cannot that internal an harbor which gis AP eetdls p150 less detected LAMP1 against ncdw,w efre muoloecnesann of staining from gene (MEFs) HPS6 immunofluorescence fibroblasts by embryonic performed of caused mouse phenotype that we a to causes knockdown, mouse similar in mislocalization function LAMP1 HPS6 of loss whether epromdlv mgn xeiet.Iaeaaye of that, analyses indicate Image vesicles experiments. mCherry-LAMP1-labeled imaging of complex, live trajectories dynein–dynactin the performed by we driven lysosomes of transport 5D). enhanced (Fig. to (EGFP) fused proten HPS6 fluorescent full-length green the in by 5C). LAMP1 rescued (Fig. also of was mice phenotype MEFs wild-type dispersal cytoplasmic of the from Moreover, those with compared vesicles eurdfrteeiula oiinn flysosomes. specifically of is positioning fragment p150 N-terminal to HPS6 bind HPS6 siRNA-resistant cannot which theperinuclear 1–700), the (aa that Flag-tagged, of overexpression for indicating by Furthermore, rescued 4D,E), required (Fig. was HPS6 LAMP1 phenotype of distribution subcellular HPS6-deficiency-induced The ed otectpamcdseslo yooe.Consistently, lysosomes. of indeed, function, dispersal of lysates HPS6 brain cytoplasmic from of immunoisolation the in loss to distributed that leads peripherally indicating both 5B), more MEFs, (Fig. were (WT) MEFs LAMP2 wild-type and to compared LAMP1 that, showed analysis ope olssmlcros ic ehv eosrtdthat demonstrated the p150 have we of motor with Since the overexpression cargoes. linking HPS6 lysosomal interacts by to whether lysosomes complex asked of HPS6 transport next retrograde we that mediates complex, and motor dynein-dynactin positioning perinuclear in lysosomes 4F,H). LAMP1 (Fig. of cells distribution HeLa subcellular the affect not however, did Flag-HPS6, of Overexpression lysosomes. of the positioning that rescue indicating not 4D,E), did (Fig. p150 4C), phenotype (Fig. dispersal HPS5 lysosome subunit the BLOC-2 other the to bind eitdb P6 ned muoloecnemicroscopy Flag-p150 of immunofluorescence overexpression Indeed, that HPS6. showed by mediated p150 elprpey(i.4,) niaigta h neato fHPS6 of p150 interaction the that with indicating 4G,H), (Fig. periphery cell eiula lseigadsraelvl eenra in normal were levels surface (Falco and from fibroblasts clustering their whereas wild-type, from perinuclear than HPS6 fibroblasts concentrated less the of are in ( compartments that, depleted mice reported been cells mutant has in It 4I). LAMP1 (Fig. with co-immunoisolated P6 osbyb optn ihedgnu p150 endogenous with competing by possibly HPS6, idn ftedni–yatnmtrcmlx eraoe that reasoned p150 we complex, this motor dynein–dynactin the of binding infcn eraei h eeso p150 of with levels lysates the in cell is decrease there significant that HeLa indicated a analysis from Immunoblot LAMP1. to lysosomes performed antibodies we of lysosomes, with immunoisolation complex motor dynein–dynactin which Flag-p50, 4G,H). (Fig. peripheral of lysosomes caused of complex, overexpression distribution protein studies 1997), dynein–dynactin the previous al., disrupts with et consistent (Burkhardt Similarly, region. perinuclear ofrhrvrf htHS,ide,mdae retrograde mediates indeed, HPS6, that verify further To aigetbihdta P6i eurdfrlysosome for required is HPS6 that established Having ots hte P6i eurdfrteascaino the of association the for required is HPS6 whether test To Glued Glued ´ n-Pe ora fCl cec 21)17 5448 doi:10.1242/jcs.141978 4574–4588 127, (2014) Science Cell of Journal bnigaiiyo P6i eurdfrteperinuclear the for required is HPS6 of ability -binding Ccue ipra fLM1lbldlssmst the to lysosomes LAMP1-labeled of dispersal caused -C ´ Glued e ta. 05 aaa ta. 06.T determine To 2006). al., et Salazar 2005; al., et rez Glued aeear pale rgetcnas lc ergaetransport retrograde block also can fragment srqie o yooa oiinn nthe in positioning lysosomal for required is Hps3 uatmc ( mice mutant Glued LC3dfcet,LAMP1-positive deficient), BLOC-3 , Ncue yolsi ipra of dispersal cytoplasmic caused -N Glued Fg A.Cnoa microscopy Confocal 5A). (Fig. Glued Glued ru cocoa ieb sn antibodies using by mice u ean h blt to ability the retains but n I nLAMP1 on DIC and Glued LC2deficient) BLOC-2 , Glued ruby-eye NbtntFlag- not but -N swl sDIC as well as ( ru Glued mice, ) Hps1 4577 for ru ru

Journal of Cell Science yaed rti ojgtdwt ihrat-P6()o niLM1()atbd.Nra g n niG10srea otos nu n on pro bound and Input p150 controls. EEA1, as serve against anti-GM130 antibodies and with IgG Normal detected antibody. and (D) SDS-PAGE anti-LAMP1 or by (C) resolved anti-HPS6 were either with conjugated G protein Dynabeads *** aeilFg 3-) are rtisfrlssmlhydrolases lysosomal for proteins carrier the S3E-H), in (supplementary Fig. late CI-MPR detected material (supplementary and was CD-MPR change of for cells distribution no subcellular Moreover, HPS6-depleted markers S3A-D). not CD63- Fig. and in material did Rab7 – of structures using – pattern distribution CD63 positive respectively the by in changes bodies, bodies. and any cells detect multivesicular multivesicular Rab7 and HeLa endosomes and against of endosomes antibodies subcellular microscopy the late for required Immunofluorescence also of is it whether distribution ask to center. us cell prompted the to lysosomes of transport retrograde the mediates EERHARTICLE RESEARCH 4578 HPS6-p150 that and complex retrograd 2). dynein–dynactin requires and 1 lysosomes Movies of indi HPS6- S2, results in Fig. these center together, material Taken cell (supplementary the towards cells vesicles depleted LAMP1 decrease of significant movement a indeed, in was, there cells, control with compared mean represent Data test. post Tukey a with variance of analysis one-way from obtained LAMP1) with (compared compartment. 10 lysosomal bar: the Scale with p230. associates HPS6 3. Fig. irgah futahnscin fDnbasfo muoslto nC ih aesaehge anfcto mgso oe ein nlf pane left in regions boxed of images magnification higher are panels Right C. in immunoisolation from Dynabeads of sections ultrathin of micrographs n rohasidct ebaossrcue soitdwt antcbas cl as 0 m(etpnl) 0 m(ih panels). (right nm 200 panels), (left nm 500 bars: Scale beads. magnetic with associated structures membranous indicate arrowheads and P htHS srqie o eiula itiuino lysosomes of distribution perinuclear for required is HPS6 That , .0) .. o infcn.(,)HS-o AP-soitdognle eeimnioae rmmmrn rcin fHL el ihmagnetic with cells HeLa of fractions membrane from immunoisolated were organelles LAMP1-associated or HPS6- (C,D) significant. not n.s., 0.001). m .()Qatfcto fHS ooaiainwt aiu akr nA seik niaesaitclsgiiac fdifferences of significance statistical indicate Asterisks A. in markers various with colocalization HPS6 of Quantification (B) m. aeta h eiula positioning perinuclear the that cate rnpr hti rvnb the by driven is that transport e Glued A eaclswr ie n muotie o P6adLM1 AP,C6,EA or EEA1 CD63, LAMP2, LAMP1, and HPS6 for immunostained and fixed were cells HeLa (A) interaction Glued sntrqie o h oiinn fLsMB,adtrafficking and TGN. LEs/MVBs, the of and positioning endosomes the between for HPS6 required Thus, not 2004). (Dı Seaman, is 2009; endosomes Klumperman, and and Saftig TGN 1998; the between shuttle that P6wt IPlbldlssmsadfudta,cnitn with consistent that, found and lysosomes LAMP1 RILP-labeled with HPS6 of of colocalization perinuclear examined and we compensatory Moreover, lysosomes. movement shift of or centripetal positioning mediate redundant material to further partially exist (supplementary that mechanisms a periphery suggesting S4A-C), cell caused Fig. the towards knockdown fluorescence double lysosomes cells, HPS6-depleted that or RILP- and showed in distributed plots peripherally more profile were functional Radial RILP is analysis. by and followed quantitative HPS6 immunostaining there by of distribution lysosome and whether knockdown examined movement and double test lysosomal performed in we To RILP positioning, and 2007). HPS6 center between al., cell redundancy the et towards lysosomes (Johansson of transport retrograde mediates M3,LM1 AP n P6 E ersnaieelectron Representative (E) HPS6. and LAMP2 LAMP1, GM130, , rvosyi a enrpre htRL id op150 to binds RILP that reported been has it Previously ora fCl cec 21)17 5448 doi:10.1242/jcs.141978 4574–4588 127, (2014) Science Cell of Journal 6 ...( s.e.m. n 5 54 el,* cells, 35–45 ´ zadPfeffer, and az P , 0.05, Glued s Arrows ls. teins and

Journal of Cell Science EERHARTICLE RESEARCH i.4. Fig. e etpg o legend. for page next See ora fCl cec 21)17 5448 doi:10.1242/jcs.141978 4574–4588 127, (2014) Science Cell of Journal 4579

Journal of Cell Science cl a:10 bar: Scale D E el from cells MEF (D) p150 EERHARTICLE RESEARCH 4580 from cells MEF in phenotype dispersal lysosome rescues HPS6 of Overexpression 5. Fig. HPS6 with colocalize not did RILP material contrast, (supplementary Rab7 In and S4D). RILP LAMP1 2001), Fig. al., et with Jordens 2001; colocalized al., et (Cantalupo reports previous ri.Iptadbudpoen eeaaye ySSPG n muoltigwt nioist p150 to or WT or antibodies WT from with from MEF immunoblotting organelles cultured and membranous Primary SDS-PAGE (B) LAMP1-associated by Myc. of analyzed and Immunoisolation were Flag (C) proteins to LAMP2. bound antibodies and and with LAMP1 Input immunoblotting against brain. and antibodies SDS-PAGE with by immunostained analyzed were were proteins bound and Input p150 eetasetytasetdwt lgvco,Fa-agdp0 p150 p50, Flag-tagged vector, cells Flag HeLa h with (G) 24 transfected LAMP1. for and transiently HPS6 Flag were Flag-tagged against antibodies with with transfected immunostained transiently and were the cells to HeLa relative (F) LAMP1 (mean of distribution D against signal in antibodies of nucleus plots with profile stained Radial and (E) siRNA- HPS6-N LAMP1. 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Fig. raelsfo eacls nu n on rtiswr nlzdby analyzed p150 were to proteins antibodies bound with immunoblotting and and Input SDS-PAGE cells. HeLa membranous from LAMP1-associated organelles of the Immunoisolation to (mean (I) relative G experiment). LAMP1 and of F distribution signal in of nucleus plots profile Radial (H) LAMP1. rp150 or AP,HS,DCand DIC HPS6, LAMP2, muosltdwt AP a eemndwt I mgJ Data ImageJ. NIH with mean determined represent was LAMP1 with immunoisolated 5 xeiet,2–2clseprmn) C E23cells HEK293 (C) cells/experiment). 20–22 experiments, 3 Glued Glued Glued rFa-agdHS n y-agdHS rMctge P6dlto uat( mutant deletion HPS6 Myc-tagged or HPS6 Myc-tagged and HPS5 Flag-tagged or n I oimnioae ihLM1wsdtrie ihNHIae.Dt ersn mean represent Data ImageJ. NIH with determined was LAMP1 with co-immunoisolated DIC and Cfr2 n muotie ihatbde gis lgand Flag against antibodies with immunostained and h 24 for -C m m. 6 .. 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Red membrane Magic a in with cells cargo incubated we delays endocytosed when depletion of HPS6 degradation that indicating the hours 6A,B), 3 (Fig. until incubation up DQ-BSA cells of HPS6-depleted weaker in cells, detected was control fluorescence with Compared 2009). Colombo, oitrc ihp150 of failed siRNA- with that mutant degradation interact truncation of the lysosomal to whereas catB, Overexpression restored MR and HPS6 lysosomal impaired. DQ-BSA that full-length was indicating resistant activity 6C,D), (Fig. enzyme cells HPS6-depleted in splmnaymtra i.SD,sgetn htte mediate they pathways. that transport suggesting lysosomal retrograde S4D), different Fig. material (supplementary a oefc Fg EH.Mroe,oeepeso fp0or p50 of overexpression Moreover, 6E-H). (Fig. effect no had esrdisfursec nest,wihi nidcto of (Va lysosomes indication in cleavage an proteolytic became we upon DQ-BSA is fluorescent endocytosed end, examined as which capacity intensity, degradation this lysosomal we fluorescence and To DQ-BSA its substrate cells. first fluorogenic measured the with HPS6-depleted cells lysosomes, HeLa al., in incubated of of activity et HPS6-mediated positioning lysosomal regulation of (Korolchuk positioning relevance flux physiological lysosomal the perinuclear the autophagic assess through that To and 2011). responses signaling found mTORC1 nutrient have coordinates studies Previous p150 with interaction its and HPS6 yooa nyeactivity and enzyme cargoes lysosomal endocytosed of degradation lysosomal ora fCl cec 21)17 5448 doi:10.1242/jcs.141978 4574–4588 127, (2014) Science Cell of Journal D 8–8)wr ye o oI ihimblzdFa antibody. Flag immobilized with co-IP for lysed were 187–189) ru mouse. Glued 6 Glued A E23clsoeepesn Flag-tagged overexpressing cells HEK293 (A) ...( s.e.m. I,LM1adHS.Rltv mutof amount Relative HPS6. and LAMP1 DIC, , u eandHS-idn ability HPS5-binding retained but n 5 xeiet,* experiments, 3 Glued r eurdfor required are P , .5 ** 0.05, ´ qe and zquez ru ru P mouse , mouse 0.01).

Journal of Cell Science nest fM aBsbtaepnt,wees h EGFP- the wherease puncta, substrate catB p150 MR of intensity EERHARTICLE RESEARCH p150 6. Fig. Glued Glued e etpg o legend. for page next See Cfso rtnddnt(i.6,) oehrteedata these Together 6I,J). (Fig. not did proten fusion fluorescence -C the in decrease a caused EGFP to fused -N o ohdgaaino noyi ag n nyeatvt of activity enzyme and cargo endocytic lysosomes. of degradation both for niaeta P6adisitrcinwt p150 with interaction its and HPS6 that indicate ora fCl cec 21)17 5448 doi:10.1242/jcs.141978 4574–4588 127, (2014) Science Cell of Journal Glued r required are 4581

Journal of Cell Science el xrsigEF,EF ue op150 to fused EGFP EGFP, mean expressing mean represent cells substrate Data catB G. MR in of knockdown ( Quantification control with (H) to incubated h. relative and 2 intensity 1–700) for (aa substrate HPS6-N catB EGFP or MR EGFP, HPS6, expressing siRNA-resistant cells knockdown to HPS6 fused or control of Snapshots (G) eaiet oto ncdw nE aarpeetmean represent ** Data cells/experiment, intensity E. 11–14 mean in experiments, DQ-BSA knockdown of Quantification control and (F) to 1–700) h. relative (aa 1 for HPS6-N DQ-BSA mCherry, or with expressing HPS6, incubated cells siRNA-resistant knockdown to HPS6 fused or mCherry control of Snapshots (E) ewe eta–8 n eta–4 Fg GH,weeswith whereas p150 7G,H), to (Fig. fused mCherry Dextran–647 and Dextran–488 between QBAi yooe.Teteto oktasetdclswt the with cells mock-transfected H of of vacuolar-type cleavage Treatment proteolytic DQ-BSA indicates lysosomes. spots with in fluorescent incubated of DQ-BSA cells appearance The knockdown h. 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Fig. ARTICLE RESEARCH 4582 or mCherry-p50 lysosomes of electron-dense overexpression and Consistently, mCherry-p150 detected lamellar-structured the 7E,F). in was decrease cells, (Fig. a no of and control lysosomes MVBs of with although number number of the compared in increase that, an size S3I), was there Fig. the revealed material in cells (supplementary difference HPS6-depleted ultrastructural fusionsignificant Furthermore, of less lysosomes. was analysis there and HPS6- that LEs/MVBs in indicating the between mCherry–LAMP1 7C,D), (Fig. with in cells depleted colocalized and decrease Dextran–488 Similarly, LEs/MVBs lysosomes. between a less of distribution fusion perinuclear was that requires lysosomes suggesting HPS6-depleted there 7A,B), in the (Fig. Dextran–647 affect However, cells and not Dextran–488 dextran. of did colocalization knockdown puncta of no HPS6 fluorescent showed that of uptake number analysis indicating and imaging 7A,B), then microscopy intensity (Fig. mean confocal Confocal were the Alexa- by 2005). in Cells to followed difference al., medium. h conjugated et 3 conjugate-free dextran (Bright for containing in (Dextran–488) medium Fluor-488 dextran chase followed in containing h h 8 12 incubated medium for an in (Dextran–647) by Alexa-Fluor-647 cells to incubating conjugated by labeled we lysosomes, decreased lysosomes and a LEs/MVBs al., the between by fusion caused et whether the is for in cells Futter failure determine HPS6-depleted in lysosomes 1997; To cargo endocytic al., 1998). to of et al., degradation cargoes Bright et 2005; Mullock endocytic al., 1996; of et delivery (Bright degradation for and required observed, was lysosomes is and lysosomes LEs/MVBs between and fusion LEs/MVBs Direct between complex fusion dynein–dynactin impair of activity disruption and depletion HPS6 oehr hs aaidct httefso ewe endocytic between fusion dynein–dynactin active the an and complex. that HPS6 requires lysosomes indicate and cargoes data these Together, nest eaiet hto oto ncdw nC aarpeetmean cells. represent 46 Data KD, HPS6 C. cells; in 49 knockdown KD, control (ctrl mean of s.e.m. substrate that catB to catB MR relative MR of intensity abolishes Quantification Bafilomycin and (D) h. B fluorescence. 2 cathepsin substrate for of substrate activation catB prevents MR treatment with incubated h cells 1 konckdown at cells knockdown control mean in represent that KD Data to HPS6 A. relative in in points DQ-BSA time of different intensity at control. fluorescence cells negative mean as of serves Quantification and (B) fluorescence DQ-BSA abolishes 1991) ( n nuae ihM aBsbtaefr2h J uniiaino RcatB mean MR represent of Data Quantification I. in (J) EGFP h. to 2 relative intensity for mean substrate substrate catB MR with incubated n n 5 5 5 xeiet,* experiments, 3 xeiet,1–3clseprmn,*** cells/experiment, 11–13 experiments, 3 xeiet,1–4clseprmn,*** cells/experiment, 17–24 experiments, 3 + Glued APs niio aioyi 1(a)(ohmr tal., et (Yoshimori (Baf) A1 Bafilomycin inhibitor -ATPase P , Ncue eraei h colocalization the in decrease a caused -N .5 ** 0.05, Glued 6 ...(tlK,3 el;HS D 8cells. 48 KD, HPS6 cells; 32 KD, (ctrl s.e.m. 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LysoSensor perinuclear that with mCherry–LAMP1 the overexpressing cells in incubated speculated place 2007; takes we al., also et Majumdar 2012), 2011; al., Mindell, et (Majumdar regulated tightly E/Vsadlssms ned ly nipratrl in role important an between plays fusion maturation. indeed, lysosomal Rab7-mediated lysosomes, that and indicating Rab7-depleted LEs/MVBs in 8I,J), fluorescence (Fig. catB was MR cells there in capacity cells, with decrease control degradative significant knockdown to a Compared lysosomal by fluorescence. detected catB cells MR enzyme We HeLa with of 8H). in (Fig. acquisition Rab7 siRNA and depleted we maturation is activity, lysosome lysosomal and for LE/MVB between /MVBs. required LEs fusion the whether of determine competence To fusion the 2009), the from regulating Ceresa, cargo by and endocytic probably (Vanlandingham of lysosomes transfer the previously the to lysosomal reported for LEs/MVBs in required been is role has that Rab7 a It that speculate plays acidification. to LEs/MVBs and/or us with maturation prompted lysosomes region of perinuclear fusion the in mature region. frequently perinuclear most the lysosomal maturation in and place acidification take between lysosome data as well lysosomes 8G), these correlation and as (Fig. LEs/MVBs Together, between fusion cells positive heterotypic positioning. that suggest perinuclear HPS6-depleted the a and near and acidification substrate peaked also control intensity catB both indicating fluorescence MR in LysoSensor mean the of nucleus its plots using profile that radial by showed the detected Furthermore, 6C,D). was (Fig. This acidification enzyme that lysosomal the lower activity the 8E,F). impaired to (Fig. contributing also thereby lysosomes, knockdown cells of HPS6 LysoSensor with HPS6-depleted that of LysoSensor indicates of intensity in colocalization the the both al., mCherry–LAMP1 in in et and decrease puncta (Lin a fluorescent 4–5 control was with of compared there Intriguingly, range cells, 1998). al., pH et Siemasko a 2001; within compartments acidified Fg A,a ela nclsta vrxrse 5 or p50 overexpressed that cells cells HPS6-depleted in and as control well both p150 as in 8A), nucleus (Fig. peaked the the Dextran–647 that and revealed near Dextran–488 plot of profile colocalization radial of the measured distance extent of 2013), its al., radial by colocalization et determined of (Niu nucleus of the centroid function of extent center a fusion the from the as starting that Dextran–647 plotted and speculate then endocytic Dextran–488 perinuclear we to the of When in us place cytoplasmic region. delivery takes lysosomes prompted both and impaired LEs/MVBs lysosomes caused between and to HPS6 lysosomes of cargoes of knockdown that dispersal fact The the in mature region and perinuclear LEs/MVBs with fuse Lysosomes nclsta vrxrse 5 rp150 or p50 overexpressed also but that 8C), cells (Fig. cells in mean HPS6-depleted The and only knockdown substrate. not nucleus control catB the in near MR peaked profile fluorescence catB of MR radial signals of intensity fluorescent with the distribution of subcellular plots and activity enzyme rb (p probe h atta yooe uewt noyi age and cargoes endocytic with fuse lysosomes that fact The iial,w netgtdterltosi ewe lysosomal between relationship the investigated we Similarly, Glued ora fCl cec 21)17 5448 doi:10.1242/jcs.141978 4574–4588 127, (2014) Science Cell of Journal K N(i.8B). (Fig. -N a 5 TM .)ta cuuae n loecsgenin green fluoresces and accumulates that 5.2) re N-8) ebaeprebepH membrane-permeable a DND-189), green Glued N(i.8) which 8D), (Fig. -N

Journal of Cell Science EERHARTICLE RESEARCH nuae ihDxrn48fr3h D uniiaino ooaiainbtenDxrn48admhryLM1i .Dt ersn mean represent mCherry-p150 Data mCherry-p50, C. mCherry, in mCherry-LAMP1 and pu Dextran-488 the between of colocalization Quantification of (B) Quantification endosomes. (D) endocytic h. of 3 labeling for allow Dextran-488 to with Dextran-488 or incubated with Dextran-488 h with labeled 3 compartments KD, for lysosome. endo-lysosomal (ctrl incubated and of LE/MVB intensity then between mean and fusion and lysosomes in number decrease label a to in Dextran-647 results HPS6 with of Knockdown 7. Fig. niaelssms ih aesaerpeettv ihrmgiiainiae fsrcue ihatpclmrhlg fMBo yooe.()Qu (F) lysosomes. or MVB of morphology mean typical represent a Data with structures E. of in images organelles magnification indicated higher of representative are panels Right lysosomes. indicate ( ihHeht332fr3 i n mgdb ofclmcocp.()Qatfcto fclclzto fDxrn48adDxrn67lbldcompa Dextran-647-labeled and Dextran-488 of colocalization of Quantification (H) microscope. mean confocal represent by imaged Data and G. min in 30 for 33342 Hoechst with n 5 5cls * cells, 35 n 5 8cls P6KD, HPS6 cells; 38 P , .5.()Rpeettv lcrnmcorpso oto lf)o P6kokon(ide el.Arw niaeMB,sldarrowheads solid MVBs, indicate Arrows cells. (middle) knockdown HPS6 or (left) control of micrographs electron Representative (E) 0.05). 6 ...( s.e.m. n 5 9cls *** cells. 39 n Glued 5 06 el,*** cells, 40–60 No mCherry-p150 or -N P , .0) C npht fcnrlo P6kokonclstasetdwt Cer-AP o 4hadthen and h 24 for mCherry-LAMP1 with transfected cells knockdown HPS6 or control of Snapshots (C) 0.001). 6 ...(tlKD, (ctrl s.e.m. P , .0) cl as 10 bars: Scale 0.001). Glued Cfr1 eelae ihDxrn67adDxrn48sqetal si ,sandlive stained A, in as sequentially Dextran-488 and Dextran-647 with loaded were h 16 for -C n 5 0 el;HS KD, HPS6 cells; 101 m (A,C,G). m 2 ora fCl cec 21)17 5448 doi:10.1242/jcs.141978 4574–4588 127, (2014) Science Cell of Journal 4 n hi ereo vra nA aarpeetmean represent Data A. in overlap of degree their and 647 n 5 0 el.* cells. 103 A npht fcnrlo P6kokonclspreloaded cells knockdown HPS6 or control of Snapshots (A) P 5 .21 *** 0.0221, P , .0) G el rnfce with transfected Cells (G) 0.001). 6 s.e.m. antification 6 rtments s.e.m. 4583 ncta

Journal of Cell Science raels ete osi fettedsrbto flate of distribution the affect these of it distribution the does on effect neither no TGN; HeLa has the organelles, in and HPS6 endosomes In involved early of is from syndrome. knockdown absent that mostly Hermansky–Pudlak is BLOC-2 HPS6 and cells, the of biogenesis subunit LRO a HPS6, identified EERHARTICLE RESEARCH 4584 of partner(s) interaction the find to out set we p150 study, this In DISCUSSION 8. Fig. Glued e etpg o legend. for page next See uui ftedni–yatncmlx We complex. dynein–dynactin the of subunit a , eut naboko ergaetasotadperipheral between and interaction the transport of cytoplasmic retrograde complex Disruption causes although HPS6-p150 lysosomes. dynein–dynactin depletion of of contrast, HPS6 the block dispersal lysosomes, In trafficking of of a distribution or TGN. activity in trafficking the the results endocytic of and on disruption endosomes of effect depletion no between Moreover, has bodies. HPS6 multivesicular and endosomes ora fCl cec 21)17 5448 doi:10.1242/jcs.141978 4574–4588 127, (2014) Science Cell of Journal Glued n h oiatngtv p150 negative dominant the and Glued N-terminus

Journal of Cell Science evsa yendnci ag dpo yitrcigwt p150 with interacting by adaptor cargo HPS6 manner. dynein–dynactin bidirectional as a in serves microtubules along catB move Lysosomes MR HPS6. of intensity mean mean of represent *** Quantification Data with (J) cell. incubated h. per cells 2 substrate knockdown for siRNA Rab7 substrate Rab7-targeting or catB of control MR mixture of a Snapshots with (I) silencing duplexes. Rab7 showing (mean cell E HeLa in nucleus the to n relative distribution signal LysoSensor el;HS KD, HPS6 cells; Cer-AP rgt nE aarpeetmean represent Data E. and in co- DND-189 (right) and LysoSensor mCherry-LAMP1 (left) between intensity overlap) mean (Mander\s min. DND-189 localization 30 LysoSensor for of mCherry- DND-189 Quantification expressing LysoSensor (F) cells with knockdown incubated HPS6 and in or LAMP1 nucleus control the of to Snapshots relative (E) substrate catB (mean MR 6I of Fig. distribution signal of plots dniidmlil yenrcutetad-euaoyfactors -regulatory and dynein-recruitment have studies multiple Previous understood. identified partially 2001). recruited only 1997; al., is are motor et lysosomes al., retrograde Jordens to the 1998; which et al., by et mechanisms (Burkhardt Harada However, 2001; lysosomes al., the for et of required Cantalupo is movement complex dynein–dynactin the centripetal and 2007) al., et r eurdfrmlnsm ignss(eodpuo tal., et that (Gerondopoulos GTPases biogenesis small melanosome two for Rab38, required and Rab32 are for organelle factor and/or exchange type-specific cell or tissue specificity/ specific. is Alternatively, cargo lysosomes provide that and/or depletion. might selectivity endosomes organelles HPS6 late lysosome-related for or by recruitment adaptors the dynein explains caused dynein–dynactin different which phenotypes trafficking, multiple lysosome mild for that exist is mechanisms that indicate identified lysosomes we these for Conceivably, study, results positioning. adaptor late this perinuclear cargo lysosome In dynein for of 2003). required another al., yet attachment et as reduced, (Falco Nazarian HPS6 the motors 2005; microtubule al., was regulates to et lysosomes microtubules BLOC-3 and endosomes LAMP1- and that along of movement LEs suggesting the organelles LAMP1-positive of cells. frequency of GFP-labeled the HeLa also clustering in but perinuclear lysosomes lysosomes the and ( only HPS1-deficient endosomes cargo dynein in – late 2011) Moreover, al., for et 2007; (Tan al., LIC1/2 adaptors et and 2001) Johansson al., 2001; et al., Jordens et (Cantalupo RILP include that inldsrbto fM aBsbtaerltv otencesi i.6C Fig. in nucleus the to relative substrate catB MR of (mean distribution 7G signal Fig. in Dextran-647 and s.e.m., Dextran-488 of (mean signals centroid colocalized the from fluorescence drawn total circle n of concentric periphery. fraction each cell the in the to present toward corresponding fluorescent extending averages colocalized and are of centroid Values distribution the the from is starting Shown signals 7A. perinuclear Fig. the in in Dextran-647 mature and LEs/MVBs with region. fuse Lysosomes 8. Fig. ARTICLE RESEARCH etrta sdie ytedni–yatncmlx(i.8K). (Fig. cell complex dynein–dynactin the specifically the towards is by lysosomes driven HPS6 is of that that transport indicates center retrograde which in effect, involved similar a has (mean ihprncerlssms cl as 10 LEs/MVBs bars: of Scale fusion lysosomes. reduced perinuclear of from with degradation resulted lysosomal possibly delays proteins, cytoplasmic it endocytosed in Moreover, resulting lysosomes. thus of HPS6 lysosomes, dispersal of of Depletion transport lysosomes. retrograde to impairs complex dynein–dyanctin the tethering 5 5 P otrcnl,BO- a dniida unn nucleotide guanine a as identified was BLOC-3 recently, Most yooe r ipre in dispersed are Lysosomes experiments, 3 experiments, 3 , .0) K oe o yooa erdto eet nteasneof absence the in defects degradation lysosomal for Model (K) 0.001). 6 n A ailpoieposo ooaie inl fDxrn48and Dextran-488 of signals colocalized of plots profile Radial (A) 5 s.e.m., xeiet,1 el/xeiet.()Rda rfl lt of plots profile Radial (C) cells/experiment). 15 experiments, 3 6 s.e.m., n n 5 5 . . experiments, 3 0cls *** cells. 40 1clseprmn) H muotiigaayi of analysis Immunostaining (H) cells/experiment). 11 1clseprmn) B ailpoieposof plots profile Radial (B) cells/experiment). 11 n 5 xeiet,1 cells/experiment). 15 experiments, 3 P , Lamp1 .0) G ailpoieposof plots profile Radial (G) 0.001). . aeear pale 1clseprmn) D ailprofile Radial (D) cells/experiment). 11 6 2 ...( s.e.m. m / m. 2 uiefbolss not fibroblasts, murine ) / Lamp2 n 6 5 ...(tlKD, (ctrl s.e.m. experiments, 3 2 / 2 el (Huynh cells 6 6 ´ Glued n-Pe s.e.m., s.e.m., n 6 ´ 5 and rez 31 uinb ncigdw a7as assardcinin reduction a causes also Rab7 LE/MVB-lysosome down blocking knocking Further, of by LEs/MVBs 8). between (Fig. clustering fusion fusion lysosomes correct LAMP2-postivie perinuclear the and for the and/or more required is the that LAMP1- lysosomes accompanying suggesting of HPS6, compartments, of distribution depleted is lysosomes peripheral cells and LEs/MVBs in between fusion impaired Ceresa, study, and our Vanlandingham In 2007; its 2009). al., protein through et vacuole (Luzio and probably complex fusion sorting) cells, (homotypic HOPS between have HeLa the fusion with in studies interaction efficient lysosomes for Previous and required function. LEs/MVBs is Rab7 and that biogenesis demonstrated lysosomal in lysosomes, such melanocytes. types, of a and whether cell fibroblasts as trafficking specialized elucidated as in serves and organelles be also biogenesis lysosome-related complex, of to the and BLOC-2 remains in the It adaptor of dynein subunit 8K. Fig. the as lysosomal illustrated in is HPS6, mediating model which in cells, the role HeLa a the in has in Our functioning it and that elusive. trafficking the reveals remain HPS6 subunits to on BLOC-2 study However, Msb3 2013). of al., function(s) et protein Peter molecular (John GTPase-activating yeast in membrane Rab5 by endosomal maturation the endosomal promote to recruiting shown was BLOC-1 2012). uinbtenLsMB n yooe rgesfurther triggers lysosomes compartment. and lysosomal the in heterotypic activation LEs/MVBs enzyme which and acidification by between mechanism(s) thus, will, investigate fusion It to 2007). al., important et Majumdar al., be 2008; et al., (Majumdar of et regulated Majumdar lysosomal tightly 2011; degradation is the acidification lysosomal lysosomal that of on activation A studies and amyloid is Previous maturation endo-lysosomes full compartment. form to for lysosomes required and fusion that LEs/MVBs notion between the supporting capacity, degradative lysosomal aoaois ecls A.Ohratbde sdi hssuyinclude study this in used anti-p150 antibodies mouse Other CA). Bio-Rad (Affi-Gel; Hercules, beads Laboratories, gel AminoLinkagarose on immobilized antigen nopCer-1adpGPC Cotc,C)frohrexperimental other for CA) (Clontech, cloned pEGFP-C1 or and assay, pmCherry-C1 pCMV- the co-immunoprecipitation into for and from Jolla) pCMV-Tag2b La PCR-amplified into (Stratagene, Tag3b were cloned and fragments construct, and pGBKT7-mHPS6 cDNA full-length HPS6 Constructs 1– (aa fragment against from His-HPS5 described antibodies recombinant purified as using polyclonal 333) raised Rabbit raised were 2004). were HPS5 al., human HPS6 et against (Gautam previously antibodies polyclonal Rabbit reagents and Antibodies METHODS AND MATERIALS H-CBoi n tetvdnaaoersn eefo Thermo from were resins MA). (Waltham, streptavidin-agarose Scientific Sulfo- antibodies and Canada). ON, secondary (Mississauga, NHS-LC-Biotin Technology (HRP)-conjugated Signaling Cell peroxidase from and were radish antibodies (Carlsbad, horse secondary Invitrogen from CA), of purchased anti- conjugates were Fluorescent goat Biotech, Alexa-Fluor-488–phalloidin anti- Cruz CA). 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Coudrier, and D. Louvard, D., Dauzonne, M.-N., Cordonnier, A. R. Spritz, and T. R. Swank, T., Suzuki, R., Gautam, N., Oiso, W., P. Chiang, ape,oewyaayi fvrac ihaTkyotts a used. was test Tukeypost a mean of as with groups described variance more are Results or of three analysis of one-way significance samples, statistical of evaluation For test). Student’s unpaired Falco upeetr material Supplementary J-J.L. to 31071175 and W.L.]. 31325017 to numbers 31230046 Natural [grant and Technology National China and the of Science and Foundation of 2014CB942802] Science Ministry and the 2011CB965002 from numbers grants [grant by supported was work W.L.This and Y.N. C.Z., L.Y., Funding The K.L., Y.N. from and help K.L. with by J.-J.L. analyzed by were written Data were was C.Z. Experiments manuscript and J.-J.L. L.Y. and K.L., W.L. by K.L., conducted by designed were Experiments contributions Author interests. competing no declare authors The interests Competing and wild-type from tissues and and cells assistance, MEF technical of for preparation ruby-eye Zhu the Ling for thank Hao critical We Zhen-Hua for manuscript. CAS) (IGDB, the Bao Wang on Shilai Tuanlao comments and and reagents, University) (Tsinghua providing Yu for Li University) colleagues (Xiamen our to indebted are We Acknowledgements seie,E.-L. Eskelinen, P., Worley, A., Lanahan, K., M. Tokito, B. V., Colomer, H., R. A. Sharp, Vallee, S., Engelender, and T. K. Vaughan, M., B. Paschal, J., C. Echeverri, Dı DiPietro,S.M.,Falco Falco M., King, S. and J. Pietro, N. Quintyne, Di D., A. Stephens, A., S. Lex, L., T. Culver-Hanlon, L. K. McCoy, and K. F. White, Higgins B., C. Hartmann, M., B. Creasy, C. Bucci, and B. B. C. Bruni, V., R. Roberti, P., Vallee, Alifano, G., and Cantalupo, T. Nilsson, J., C. Echeverri, P. K., J. J. Luzio, Burkhardt, and J. M. Gratian, A., N. P.Bright, J. Luzio, and M. B. Mullock, J., B. Reaves, A., N. Bright, J. V. Allan, References at http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.141978/-/DC1 online available material Supplementary infcn at significant a,E n ffe,S R. S. Pfeffer, and E. ´az, 20) h emnk-ulksnrm HS)adHS rtisare proteins biogenesis the HPS4 in organelles. and involved lysosome-related BLOC-4, (HPS1) and of BLOC-3 1 complexes, two syndrome of components Hermansky-Pudlak The (2003). itiuinaddnmc fLm1cnann noyi raelsin organelles endocytic Lamp1-containing BLOC-3. of in deficient dynamics fibroblasts and Distribution n autophagy. and dynactin. of subunit p150Glued the A. with C. interacts Ross, and L. E. Holzbaur, mitosis. during organization for Biol. spindle function and Cell reveals alignment J. dynactin chromosome of in subunit complex 50-kD the the of characterization Molecular trafficking. receptor phosphate endosomes. on trafficking protein facilitate to C. complex E. Hermansky-Pudlak Dell’Angelica, the and G. containing HPS6. complex and HPS5 a HPS3, proteins BLOC-2, syndrome of Characterization microtubules. along skating by processivity J. S. subpopulations. cell live in measure activity to cathepsin staining cysteine immunofluorescence and substrates fluorogenic using of assay movement the for microtubules with cooperate alpha lysosomes. I myosin and filaments to transport for required effector Rab7 the lysosomes. (RILP): protein lysosomal interacting disrupts complex distribution. dynactin 139 organelle the membrane of of subunit maintenance cells. (p50) dynein-dependent living dynamitin the in of Overexpression events kissing and fusion Biol. concurrent Curr. by mediated resultant lysosomes the from re-formed are and endosomes organelles. late hybrid with fuse can lysosomes n-Pe ´ 469-484. , 20) irtbl-idn oani yatnicessdynein increases dynactin in domain microtubule-binding A (2006). ora fCl cec 21)17 5448 doi:10.1242/jcs.141978 4574–4588 127, (2014) Science Cell of Journal 5B/Jmice. C57Bl/6J rz .M,Nzra,R,Sbti .adDl’neia .C. E. Dell’Angelica, and C. Sabatti, R., Nazarian, M., J. ´rez, 21) yolsi dynein. Cytoplasmic (2011). 15 o.Bo.Cell Biol. Mol. J. EMBO 132 P 360-365. , , 20) oe fLM- n AP2i yooebiogenesis lysosome in LAMP-2 and LAMP-1 of Roles (2006). 617-633. , o.ApcsMed. Aspects Mol. .5 * 0.05. ´n-Pe .Cl Sci. Cell J. t ts rnnaaercStudent’s nonparametric or -test 20 rz .M,Tna . et,S . ak,M . Raposo, S., M. Marks, R., S. 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Journal of Cell Science i . hn,Q,Os,N,Nvk .K,Gua,R,OBin .P,Tnly .L., C. Tinsley, P., E. O’Brien, R., Gautam, K., E. Novak, A., N., Oiso, E. Q., Zhang, Roberts, W., Li, H., F. Siddiqi, M., Lichtenberg, S., Saiki, I., V. Korolchuk, A. T. Schroer, and J. S. King, le,D . oa,R,Ia,V,Mrym,K,vnVimn . uty N., Murthy, T., Vlijmen, van K., Moriyama, V., Ivan, R., Rojas, P., D. Kloer, L., Janssen, S., Dusseljee, M., Marsman, M., Fernandez-Borja, I., Jordens, onPtr .T,Lcmn,J,Rn,M,Bne . arr,M and M. Cabrera, M., Bunge, M., Rana, J., Lachmann, T., C., A. Kuijl, Peter, L., John Janssen, I., Jordens, W., Zwart, N., Rocha, M., Johansson, un,K . seie,E . ct,C . aeaes . atg .and P. Saftig, A., Malevanets, C., C. Scott, L., E. Eskelinen, K., K. Huynh, A. Helenius, and J. Huotari, Zeeuw, De R., B. Dortland, A., S. Howell, A., Akhmanova, C., C. Hoogenraad, aaa . ae,Y,Kni . aaa . oaa .adHirokawa, and S. Nonaka, Y., Tanaka, Y., Kanai, Y., Takei, A. A., F. Barr, Harada, and A. Linford, R., J. Liang, L., Pietro, Langemeyer, Di A., K., Gerondopoulos, E. Novak, J., Tan, Q., Zhang, W., Li, S., Chintala, R., Gautam, R. C. Hopkins, and J. L. Hewlett, A., Pearse, E., C. Futter, ARTICLE RESEARCH 4588 J. J. Liu, and X. Zhao, K., Li, Y., Niu, C., Zhang, Y., Yang, Z., Hong, i,H . emn . ag .S n aoiz .R. J. Lakowicz, and S. J. Kang, P., Herman, J., H. Lin, ui,J . ro,P .adBih,N A. N. Bright, and R. P. Pryor, P., J. Luzio, audr . rz . sma,N,Bxam . oa,I,Lbl .and P. Lobel, I., Sohar, A., Buxbaum, N., Asamoah, D., Cruz, A., Majumdar, ulc,B . rgt .A,Fao,C . ry .R n ui,J P. J. Luzio, and R. S. Gray, W., C. Fearon, A., N. Bright, M., B. Mullock, A. J. Cooper, and D. Sept, K., J. Moore, A. A. T. J. Schroer, and Mindell, M. Rodgers, E., J. Godfrey, C., K. Maier, A., K. Melkonian, H. Kanazawa, and H. Inoue, N., Nakamura, S., Tanaka, M., Matsushita, S. J. Bonifacino, G. and K. Raposo, Moriyama, A., and J. F. Martina, H. Heijnen, S., M. Marks, R. F. Maxfield, and K. G. Gouras, D., and Cruz, P. 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Galjart, and dynein-dynactin F. 3500. the Grosveld, complexes. in these P., with functions interacting Visser, by Bicaudal-D2 pathway R., Golgi-associated Willemsen, Mammalian I., C. ermrcmoetSX neat ihdnci 10Gud n mediates and p150(Glued) dynactin with transport. interacts endosome-to-TGN SNX6 component retromer Rab32/38 dynein. a cytoplasmic is syndrome N. Hermansky-Pudlak factor. in exchange lysosome- nucleotide mutated of guanine biogenesis BLOC-3 the (2012). of component a (BLOC-2). is complex-2 T. organelles R. protein related Swank, (cocoa) and 3 C. syndrome E. Dell’Angelica, M., S. and lysosomes. mature with complexes directly receptor fuse EGF-EGF then internalized containing endosomes ieiecaatrzto fnvllwp probes. low-pH novel of characterization lifetime biogenesis (BLOC-1). the 1 of complex member a organelles dysbindin, lysosome-related mutant of from results (HPS-7) 7 type syndrome function. erdto fAzemrayodfibrils. amyloid Alzheimer of degradation R. F. 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