oeua elBooy nvriyClee odnW1 B,UK. 6BT, WC1E London College, University Biology, Molecular Ato o orsodne([email protected]) correspondence for *Author UK. 2AZ, SW7 London College, Imperial erfs eia imn pteimb A n PMEL and Burgoyne OA1 Thomas the by in epithelium movement pigment and retinal shape zebrafish number, melanosome of Regulation ARTICLE RESEARCH ß 1400 2015 February 9 Accepted 2014; October 10 attributed. properly Received is work original the Attribution that Commons provided Creative medium the any of distribution in terms use, reproduction the unrestricted and under permits distributed which article (http://creativecommons.org/licenses/by/3.0), Access License Open an is This 1 associated harmful are 2005). during movement and (Strauss, reduce arise biogenesis segments melanosome that in outer can radicals Defects photoreceptor free melanosomes which of remove with phagocytosis and and pigment, packed light densely backscattered segments 2005). is (Strauss, outer RPE containing cycle visual the shed the Additionally, in RPE phagocytosing photopigment The removing regenerating products, retina. and nutrients the providing waste to photoreceptors, outer blood the the maintains bringing of layer between a capillaries choriocapillaris, lies the fenestrated and (RPE) photoreceptors the epithelium of segments pigment retinal The INTRODUCTION pigment Retinal epithelium Melanosome, PMEL, OA1, WORDS: KEY in localisation on depends processes. that apical RPE the the in function a controls shape melanosome melanosome apical that and that regulated the independently are indicate shape enter and findings number melanosomes cylindrical our together, only Taken but processes. body cell the in co-exist volumes melanin similar apical containing melanosomes the cylindrical and into Spherical integrity. movement photoreceptor of melanosome maintenance for and processes required is turn in zebrafish, in which melanosomes cylindrical of generation the for required biogenesis,is show melanosome mammalian of further component key We a PMEL, that formation. macromelanosome melanosome reduced , precede where disease numbers ocular mammalian a the human recapitulating number, of of feature melanosome key in form reduction common major a most induces the of cause homologue human which the in molecular mutations GPR143), the as facilitates known (also in OA1 biogenesis because of melanosome knockdown process Morpholino-mediated morpholinos. of using this manipulation timing studying the for short system providesRPE zebrafish a model the in ideal that show predominantly an we occurs Here, window. it time because embryonic challenging is epithelium pigment (RPE) retinal the in biogenesis melanosome of Analysis ABSTRACT C nttt fOhhlooy odnE1 E,UK. 9EL, EC1V London Ophthalmology, of Institute UCL 05 ulse yTeCmayo ilgssLd|Junlo elSine(05 2,10–47doi:10.1242/jcs.164400 1400–1407 128, (2015) Science Cell of Journal | Ltd Biologists of Company The by Published 2015. 1 ai .O’Connor N. Marie , 2 R aoaoyfor Laboratory MRC 1,2 iulC Seabra C. Miguel , 3 NHLI, ndvlpetbfr h nrae eaooesz (Incerti size melanosome increased the early of before reduced In number is development defects. al., number reduced visual in et melanosome with a mouse, d’Addio associated OA1-knockout by 1995; is the characterised and al., melanosomes is et enlarged disease (Bassi OA1 This GPR143) receptor, 2000). as coupled protein known G (also heterotrimeric the of established. mutation fully of be regulation to molecular of remains the movement and melanosomes macular and effects RPE of biogenesis the roles age-related their in protective precise movement and the their potential established, and aging well the are with in melanin pigment associated Although melanin is of degeneration. deficit RPE a and the disease, retinal human with per oocri amla P Fte ta. 04,the 2004), al., et al., (Futter biogenesis et RPE actin melanosome Wu mammalian 2001; of in the al., process occur et to similar with Wu appears a 2001; Although interaction to al., 2002). et capacity Rab27a-dependent (Hume the gains a process a in requiring , the neighbouring to visible, melanosomes fibrils transfer longer which in no al., IV, II) et stage are Raposo to 2001; maturation (stage fibrils al., subsequent et the During (Berson immature 2001). upon melanosomes Melanin- III melanin stage the of 2008). form to deposition to al., the catalyse delivered et melanosome of (Hurbain enzymes elongation the promotes melanosome across synthesising extend process immature and this expand melanosome, the fibrils the mammalian the of As during length 2006). al., elongation (Hellstro et melanocytes Theos melanosome in 2013). biogenesis al., for to melanosome et shown Watt required been 2011; previously be al., has an et formation forming Niel fibril vesicles polymerises, PMEL-dependent (van is and matrix intraluminal processed fibril melanin is amyloid onto which PMEL PMEL where sorting of upon (ILVs) fragments fibrils processing, CD63-dependent Proteolytic 2001). of undergo al., proteolytic et generation (Berson undergoes the deposited pigmented-cell- cultured to PMEL, a leading melanosomes), on protein, I from studies (stage specific derive 2001). MVBs al., From to those et Within shown (Raposo 1963). (MVBs) been /bodies al., multivesicular have in been melanosomes et performed have melanocytes, (I–IV) been (Seiji maturation of have described stages biogenesis four where of melanosome majority melanocytes, the culture on reason, cell this RPE For studies melanosome of melanosomes. for scarcity make melanosome window the that and time other systems life short and embryonic the in OA1 is biogenesis of RPE hampering the function individual Also in in the dissect. regulators OA1 to of of difficult elucidation role RPE the the the in made and steps has size trafficking 2008) roles - and al., of et multiplicity regulates number its (Palmisano also movement melanosome OA1 melanosome that dependent regulated. suggesting independently 2000), are al., et h otcmo omo clrabns scue by caused is albinism ocular of form common most The 3 ailF Cutler F. Daniel , 2 n lr .Futter E. Clare and me l,2011; al., et ¨m 1, *

Journal of Cell Science ln h pclpoessi uhmr xesv hnin than extensive of aspect more this much of analyses is for opportunities processes providing apical mammals, apical movement the the light-dependent to this along outer , access photoreceptor and allows the with that In contact segments. 2004) intimate in al., are et that processes Gibbs undergo 2004; (Futter cytoskeleton al., however, actin et do, the with melanosomes interaction RPE RPE Rab27a-dependent postmitotic life. largely the throughout in cells retained are melanosomes mature ARTICLE RESEARCH ecudfn eysmlrpoie nmmainREcells RPE 2; mammalian (Fig. in ILVs However, profiles S2). RPE. similar of Fig. zebrafish very material lumen (supplementary find in the could S2) to we Fig. III of appeared material suggestive stage sometimes supplementary been ‘holes’, melanosomes and has mature contain melanin melanosomes melanin) which The II upon without deposited). stage fibrils immature fibrils (containing melanosomes identify visible 2). to (Fig. melanosomes (containing unable and IV melanin stage were with mature We packed be densely to were majority appeared vast dpf therefore the 2 biogenesis, at melanosome melanosomes intense of this mature of dpf be spite 2 to In at appear RPE melanosomes the most in but occurs production melanosome to Intense due efficacy size. MO in of increase dpf, larvae loss the 5 the as to By dilution owing with distribution. presumably treated and MOs, larvae in OA1 size recovered largely melanosome had numbers in melanosome to defect prior birth latent at This numbers in a melanosome 1). reduced reduction show (Fig. to have significant dpf found mice been 2 OA1-knockout a where at was studies number mammalian there corroborates therefore, larvae and, but area zebrafish controls, melanosome MO-treated of but that OA1 compared distribution with or dpf of size material melanosome 2 in (supplementary analysis difference no dpf showed at TEM 5 at S1). control-morpholino- pigmentation clear Fig. and less could reduced was targeting OA1 embryos OA1 of their treated between depleted difference by Embryos the 1). identified (Fig. number be dpf melanosome 5 be to there in and that dpf increase 2 indicates 5 fivefold) melanin than between and size of (greater 2 large melanosome area a the in at is ages, in increase RPE two small thresholding the these increase a between in Image huge for melanin dpf. Correcting a 5 of quantified. is area and there the 2 allowed that between production embryos entire the melanin and zebrafish was through fixed of sections It were ultrathin retina (TEM). larvae from evaluate microscopy apparent (dpf) electron immediately to post-fertilisation transmission days for for (MOs). and 5 transcript processed morpholinos in and the antisense cells 2 disease, with biogenesis human At targeted mammalian study was melanosome to OA1 in model zebrafish of a that as regulation zebrafish with the zebrafish compare size To dpf melanosome 2 increased at causing number without melanosome reduce between morpholinos OA1 relationship novel a RESULTS reveal zebrafish and movement. the and system of biogenesis utility model melanosome potential a OA1 the key cells, demonstrate as two mammalian to on in in PMEL, focus defects biogenesis and We of melanosome disease. role melanosome of retinal the regulators human of and in RPE regulation processes the these molecular in movement the and biogenesis determine to which 1979). Laties, and (Burnside behaviour their nti td,w sestezbaiha oeta oe in model potential a as zebrafish the assess we study, this In ihnteMBsrcue,rte hno irl Fg 3; serial to or (Fig. Utilising melanin section. difficult the by fibrils of depth was surrounded the on ILVs into it immature running were fibrils than Initially PMEL spots the these S3). rather whether Fig. discern structures, spots Within material immature dense MVB as supplementary made identify. deposited the melanin be to within to of appeared melanin amounts effective, melanosomes, embryos easy less RPE. small becoming the was the of melanosomes MO in the presence identified of II when readily the stage dpf, be 5 fibrillar colour by dpf, not However, 2 still at skin could but, melanosomes S1) diluted Fig. material (supplementary through identified inhibit to used were MOs maturation. tyrosinase are melanosome zebrafish, simulate To in they 2007b). model of al., as similar et (Lopes a numbers, II stage inhibition melanosome beyond mature a immature to unable causes that in transiently increase have synthesis, RPE the major melanin We tyrosinase, for of mammalian enzyme lived. deletion rate-limiting functional short by in maturation very melanosome found are rapid so melanosomes previously be might immature maturation melanosome that that suggested dpf 2 melanosomes at immature identifiable morphologically of absence The RPE the in immature melanosomes potential reveal morpholinos Tyrosinase yidia eaooe a esta hto spherical of of that diameter than The less melanin. spherical they was of that melanosomes and amounts indicating cylindrical volumes, similar Cylindrical contained similar and contained very segments. dpf 3D 5 had at their melanosomes alongside outer present and parallel were aligned dpf photoreceptor but 5 melanosomes dpf processes cylindrical and 2 Apical exclusively 2 at body. cell absent at rounded the were present in with arbitrary diameter) were appeared core orientation types in constant Both were, a melanosomes ends. (with elongated cylindrical the 4). dimensional that (Fig. fact, melanosome, three showed melanosomes ‘elliptical’, analysis were of as (3D) elongated sections types described thinner previously distinct TEM Although and two spherical serial revealed and larger dimensions, shape This melanosome three the examined. elucidate in the To were sections. orientation melanosome this thin the If of in interpretation membrane. inaccurate shape with basal an infer oriented the could were with it dpf case, most parallel with 2 that axis arrangement long conceivable at tight compared the the is melanosomes to it RPE Owing of melanosomes, 2). of (Fig. zebrafish majority spherical be the in to appeared and packed cells, Melanosomes densely mammalian elongation. very melanosome previously promote are has to melanosomes shown processes immature been apical within the elongation enter cell ones Fibril RPE cylindrical the only in but co-exist body fibrils melanosomes of the populations Two to perpendicular S2). sectioned Fig. material dense melanosomes (supplementary as appears in RPE mouse spots in in melanin melanosomes that noteworthy fibrillar is immature deposited It melanosomes S3). III been the Fig. stage had material resembled to these (supplementary melanin so and parallel which discerned, be sectioned on could on fibrils was occasions axis, melanosome rare deposited longitudinal the immature is on an melanin Indeed, when melanosomes. RPE, has immature view mammalian as in of that, were fibrils in plane suggesting S3), described spots the Fig. to been material melanin perpendicular supplementary dense 3; running (Fig. fibrils the as microscopy, revealed electron section ucsflM-eitdtrsns elto a readily was depletion tyrosinase MO-mediated Successful ora fCl cec 21)18 4010 doi:10.1242/jcs.164400 1400–1407 128, (2015) Science Cell of Journal 1401

Journal of Cell Science EERHARTICLE RESEARCH 1402 have cells mammalian in studies (supplementary PMELa Deletion than S4A). regions Fig. domain) material (PKD) (repeat disease (Schonthaler kidney RPT polycystic PMELb, shorter and and has PMELb PMELa express 2005). termed al., zebrafish et PMEL, shape RPE, of melanosome mouse forms and on RPE two human MOs mammalian Unlike PMEL of analysed. of that was effect reflecting the manner a melanosomes, melanosomes in cylindrical PMEL of requires generation the melanosomes whether higher cylindrical determine of To formation the a inhibit MOs PMELa had melanosomes cylindrical ratio. surface:volume and melanosomes, htPEai atclri eurdfrtegnrto of generation the for required is implies zebrafish. particular This in melanosomes S4B). in cylindrical Fig. PMELa material of MO-treated MO- the PMELb supplementary that loss the PMELa&b 5; in of (Fig. and evident preferential less larvae PMELa was a circularity which the larvae, was the treated in there melanosomes Measuring PMELa that cylindrical small of 5). revealed a (Fig. knockdown melanosomes was after PMELa&b There number 5). and melanosome (Fig. dpf in 2 decrease at morphology melanosome their 2013). in and al., fibrils et of (Leonhardt appearance the periodicity in region RPT the implicated ML n ML& O asdacerdsuto of disruption clear a caused MOs PMELa&b and PMELa ora fCl cec 21)18 4010 doi:10.1242/jcs.164400 1400–1407 128, (2015) Science Cell of Journal eaooe aea perneo hls (D). 1 ‘holes’ bars: of Scale mature appearance Some an dpf. have 2 melanosomes images at High-magnification melanosomes (C,D) mature (B). of dpf dpf 5 2 and between (A) number large melanosome a in show increase RPE zebrafish dpf. of 5 micrographs and 2 mature between of melanosomes biogenesis Intense 2. Fig. erfs.K,kokon cl as 20 bars: Scale knockdown. MO-treated KD, OA1 zebrafish. (C,D) and from control shown (A,B) are Images melanin. electron- dense only highlighting cross-sections zebrafish eye of size. images melanosome Contrast-enhanced affecting (A–D) without number dpf melanosome 2 reduce at MOs OA1 1. Fig. n p.Rslsso h mean the show * 2 Results at dpf. diameter 5 melanosome and had on MO effect OA1 apparent The no (F) and . controls MO-treated between OA1 significant area no melanin in in resulting appears difference effective, MO less OA1 the be dpf, to 5 MO- By in OA1 zebrafish. reduction and treated significant controls a between is area there melanin dpf 2 At (E) P , .5(Student’s 0.05 m AB,10n (C,D). nm 100 (A,B), m t -test). 6 s.e.m.; Electron m m.

Journal of Cell Science EERHARTICLE RESEARCH ftrsns,wihpeet eai ytei Lpse al., et (Lopes synthesis function melanin of early prevents loss of which by fibrils identification facilitated tyrosinase, the upon is of RPE, than melanosomes mouse II) rather In (stage is MVBs, 1975). fibrillar melanin al., of zebrafish, et in ILVs melanosomes, melanosomes, (Turner that, the mature proposal immature around in the ‘holes’ deposited to fibrillar led of previously presence of has the with absence identify. together to difficult apparent were melanosomes The immature fibrillar dpf, 5 Our activity. size stage. OA1 melanosome by later of regulation number a independent but and at the birth disease. support develops knockout at thus only OA1 reduced data size are the human with melanosome numbers with treatment melanosome increased obtained the which following results in to affected mouse, reduced in similar not MOs, the was OA1 observed recapitulates most size albinism the Melanosome numbers ocular simulate of to melanosome RPE. cause MOs the are OA1-targeted common in MOs biogenesis using injected of in melanosome study Furthermore, when the of number for dpf, mechanisms organism melanosome 5 model molecular a in and as (Lopes zebrafish increase 2 suggests models effective, between huge mammalian RPE The in zebrafish study 2007b). to al., renders process et biogenesis difficult melanosome RPE a for this window time short The DISCUSSION zebrafish. affected MO-injected less PMELb also the movement were in and segments outer shape photoreceptor melanosome phenotype, less-severe observed. a segments outer with damaged Consistent or lost outer with photoreceptor integrity, present. affected segment clearly are MOs segments PMELa&b outer and photoreceptor PMELa immature dpf, PMELa 5 at upon but, dependent is that cells RPE expression. within shape apical the melanosome position the larger between to and link in the access in clear gain that a melanosomes to demonstrating but, appeared able processes, of not it were 4C) Therefore, loss melanosomes melanosomes 6). spherical Fig. mostly (Fig. significant RPE processes were material was dpf, apical 5 larvae supplementary there at MO-injected addition, 6; dpf, (Fig. PMELa&b 2 and at spherical PMELa results the in to morphology photoreceptor Similar affect and the processes into apical movement melanosome prohibit morpholinos PMELa t2df htrcpo ue emnshv o e formed yet not have segments outer photoreceptor dpf, 2 At ept h nes eaooeboeei ewe and 2 between biogenesis melanosome intense the Despite oee,hr,sra eto nlssrvasta these that 1975). running reveals fibrils al., melanin-coated analysis et fact, in section (Turner are, melanin goldfish serial of speckles in here, melanosomes, previously However, ILVs immature on described melanin resembled of that as appearance identification speckled MOs, a tyrosinase had the which using zebrafish, facilitated in also approach similar A 2007b). oto fmlnsmshsirglrmlnndpsto and deposition large melanin a matrix, irregular fibril has PMEL melanosomes a without of addition, portion In (Hellstro mice 2011). in al., inactivation PMEL et of melanosomes, phenotype cylindrical the of to number fibrils similar causes the PMELa PMEL in zebrafish reduction of of significant packing a Knockdown 2013). al., the al., et regulates et (Schonthaler domain (Leonhardt RPE RPT the The in 2005). Additionally, expressed PMEL. exclusively mammalian is longer and a PMELb has PMELb and than melanocytes domain and RPE RPT the in expressed is simply PMELa to likely not two respectively. pheomelanosomes, the are and eumelanosomes that spherical, represent suggesting and 2011), cylindrical al., populations, et most (Biesemeier and been eumelanin that pheomelanin of mixture indicated a has contain have melanosomes RPE studies which mammalian microscopy but 1984; that pheomelanin, electron and study, eu- present analytical between al., like the distinguish to In possible 1975). et not somewhat al., was et it (Turner (Jimbow fish 1979), in occur shape to al., proposed red/blonde a elliptical et the in might than results Jimbow which but that rather characterised matrix in vesiculoglobular well spherical way a less on The deposition is involve 1963). Seabra, deposited and al., is (Marks pheomelanin et melanosomes Seiji elliptical leading of 2001; fibrils, of The formation PMEL 2004). the upon carrier al., to deposited that et volume is (Slominski production eumelanin indicated similar pheomelanin black/brown the versus from have a eumelanin arise of ones, can studies of morphology cylindrical melanosome reshaping Previous the melanosomes distinct of a content. spherical precursor are melanosomal a the not rather of are but they volume is that one similar suggests RPE; greater The but the spherical. is in other diameter the melanosomes whereas cylindrical, of and microscopy narrow electron populations section two serial but revealed elongated, be to the appear of not orientation are the of melanosomes melanosomes. because immature packed sections densely thin in within very in that see fibrils therefore, to appears, RPE difficult It view. zebrafish of the plane the to perpendicular ncnrs ommas erfs xrs w om fPMEL. of forms two express zebrafish mammals, to contrast In did body cell RPE zebrafish the in melanosomes the of Many ora fCl cec 21)18 4010 doi:10.1242/jcs.164400 1400–1407 128, (2015) Science Cell of Journal eaooe cl as 0 nm. 100 bars: the Scale of melanosome. thickness entire the running through fibrils right-hand indicate the arrowheads in shown panel; slice the of position indicate the lines Dotted running melanosome. fibrils the reveals through data micrograph rendering the 3D (F) of tyrosinase dpf. 5 a at from zebrafish cell MO-treated RPE an in immature melanosome same the of micrographs fibrils. contain that melanosomes immature RPE reveals MO-treated tyrosinase microscopy of electron analysis section Serial 3. Fig. AE eilsection Serial (A–E) 1403 ¨m

Journal of Cell Science EERHARTICLE RESEARCH ihtels fclnrclmlnsmswe ncigdown knocking when 1404 melanosomes cylindrical of together loss The melanosomes, the deposition. immature with within eumelanin fibrils and of demonstration shape melanosome (Hellstro PMEL-dependent cylindrical between hair relationship the potential a in supporting content 2011), eumelanin 40– in a reduction to and leads 50% mice formation in RPE inactivation in PMEL amyloid formation biogenesis. fibril melanosome in regulate role lesser a together in and imply could packing, RPT fibrillar differences which smaller the reflect regions, Alternatively, could RPE. PKD the PMELb in and levels expression PMELa of observed effects of the is in differences knockdown effect These PMELb. smaller of knockdown A following morphology. melanosome uneven me al., et ¨m eetbei h P elbd nyclnrclmelanosomes cylindrical only body cell RPE the in detectable serial that found we RPE zebrafish as in fibrils section, melanosomes. to visualise to of appropriate necessary plane was be analysis the section not in might fibrils melanosomes visualise the of orientation Alternatively, deposited. the is subset been pheomelanin a which represent previously might in 1975) melanosomes have al., of et that (Turner melanin MVBs RPE fish of in of described ILVs suggestive the around profiles deposition Conceivably, RPE. and zebrafish in mammalian manner similar a in of occurs biogenesis model melanosome eumelanin the that evidence substantial provides PMELa, lhuhbt peia n yidia eaooe were melanosomes cylindrical and spherical both Although n yidia eaooe aesmlrvlms eut show Results volumes. mean similar the have melanosomes cylindrical cylindrical a 1 and and bars: Higher- (G) Scale (G,H) melanosome (H). cylindrical dpf. spherical melanosome Only 5 a (E,F) at of dpf. processes images 5 apical magnification at the are body in melanosomes cell are spherical RPE melanosomes and the Cylindrical in RPE (C,D) packed the dpf. densely in 2 found at are body melanosomes cell spherical and generate melanosomes. cylindrical (cyan) to Both cylindrical used and (A,B) images (green) spherical section of serial (B,D,F) of models stack a only processes. from but apical micrographs body, the cell enter RPE two ones the reveals cylindrical in analysis melanosomes microscopy of electron populations section Serial 4. Fig. ora fCl cec 21)18 4010 doi:10.1242/jcs.164400 1400–1407 128, (2015) Science Cell of Journal 6 s.e.m. m AF,10n GH.()Spherical (I) (G–H). nm 100 (A–F), m ACE igeelectron Single (A,C,E)

Journal of Cell Science EERHARTICLE RESEARCH erfs ooo fmoi Ia y7a ontdslya display not RPE do the fish myo7aa, of with VIIa, mutants associated myosin However, of be 2004). homolog to al., zebrafish found et 2014), (McNeil been al., melanosomes et has King-Smith associated Rab27a 2004; tightly al., are and oriented et that (Futter uniformly processes melanosomes of apical with bundles the have in filaments also Lopes 2004; actin Teleosts actin- al., 2007a). et the Gibbs al., 2004; to al., et the et body the (Futter cell processes RPE, apical moves microtubule-rich rich mammalian of the transfer that from In the melanosomes large regulates machinery processes. complex the the molecular Rab27a–MyRIP–myosin-VIIa for within Additionally, surface the greater melanosome a 2004). with provides cylinder al., interaction a of et ratio the surface:volume accommodate (Futter to order in distend melanosome likely must which diameter, processes, of the apical membrane narrow diameter the facilitate at narrow the the curvature within could with movement high facilitates round- together The melanosome the processes. apices, the apical which melanosome the in of into ways shape movement of and cylindrical number shape these a melanosome ended together, are between Taken There relationship inhibited. distribution. a also reveal was into findings movement processes MOs, apical cylindrical PMELa of the using generation inhibited the was When melanosomes processes. apical the entered lyasrcua oei uprigteotrsget,arl that role effect. a segments, might outer this the processes supporting explain in apical role structural the could a play within light melanosomes protection backscattered reduced Additionally, hence, The harmful and, defects. body melanosomes apical photoreceptor from apical cell the the RPE of in of affected cause loss melanosomes primary less the of is loss much processes that the outer suggests apical with the melanosomes, photoreceptor in compared melanosomes affected of loss processes, complete melanosomes almost overall the RPE reduced segments, that microscopy. of possibility electron the section numbers exclude thin were cannot in we which disrupted Although segments, or outer missing photoreceptor either affects also but RPE of the absorption to maximise area to segments surface scatter. outer light greater the the a of to surfaces access presenting lateral light the while to maximising segments, area thus surface outer light, smaller incoming a of presents it direction sphere, localisation. a its with regulating Compared to addition II in consequences, myosin functional including proteins 2007). motor King-Smith, other and (Barsoum of suggesting 2014), involvement al., et the (Wasfy movement melanosome in defect ncigdw ML o nyafcsmlnsmso the of melanosomes affects only not PMELa down Knocking direct has likely melanosome the of shape cylindrical The ora fCl cec 21)18 4010 doi:10.1242/jcs.164400 1400–1407 128, (2015) Science Cell of Journal ML C n ML& O D.Saebars: Scale (D). MOs 1 PMELa&b (B), and PMELa (C) (A), PMELb control for with dpf injected 2 zebrafish at morphology melanosome highlighting MO. extent PMELb greater than of a number to the melanosomes reduces cylindrical MO PMELa 5. Fig. ML O eut hwtemean the * show the Results than MO. shape PMELb melanosome a on had effect PMELa greater significant melanosomes. a cylindrical in of resulted loss MOs PMEL Ctrl, All melanosomes. (F) of control. number the in decrease P m , .()TePEabM eutdi a in resulted MO PMELa&b The (E) m. .5 ** 0.05, P , .1 *** 0.01, AD lcrnmicrographs Electron (A–D) P , .0 (Student’s 0.001 6 s.e.m. t -test). 1405

Journal of Cell Science EERHARTICLE RESEARCH ubradsaerlt oterfnto so increasing (at of 1406 size is melanosome changing size, function without movement altered their depletion, be can OA1 number to how melanosome through and RPE, relate zebrafish Additionally, the In shape interest. biogenesis RPE. and functional the extensive and number melanosome in mechanisms the ideal molecular specifically of an of and be study will significance biogenesis the this for RPE, melanosome zebrafish model in Given of movement RPE. the melanosome of timing processes apical the melanosomes the access of to shape necessary PMEL- is cylindrical that the the that the generate and fibrils zebrafish to dependent in contribute roles similar RPE, perform biogenesis the in as degeneration. also well retinal within are observed as defects Rep1 trafficking degenerate. photoreceptors, and so and the VIIa gradually photoreceptors myosin the in both also expressed PMEL, photoreceptors Rep1, unlike and the However, VIIa myosin into the of movement function of respectively, melanosome loss to in due processes defects and apical 1996), 1999) involve Seabra, al., also 1998; et spherical Liu al., packing which a 1998; et versus al., (MacDonald melanosome et cylindrical (Liu X-linked a increased syndrome by Usher the achieved In shape. be by mean can the that show facilitated density Results processes. be apical the bars: in would Scale melanosomes) asterisks. fewer the (thus by PME melanin shown (A), as of control segments area with outer reduced injected disrupted a zebrafish or in *** missing for morphology. resulted in dpf photoreceptor MOs resulted 5 on MOs PMELa&b at effect PMELa&b and processes profound and apical PMELa a the (B,D) has in (D). and 4 positioning MOs processes melanosome PMELa&b apical on and the effect (C) into the PMELb showing movement micrographs melanosome Electron prevents (A–D) MO PMELa 6. Fig. m P ehv hw htkymmainrgltr fmelanosome of regulators mammalian key that shown have We .()Pooeetr nfs netdwt ML n ML& O a infcnl ee yidia eaooe t5df tl oto.()PMELa (F) control. Ctrl, dpf. 5 at melanosomes cylindrical fewer significantly had MOs PMELa&b and PMELa with injected fish in Photoreceptors (E) m. , .0 (Student’s 0.001 t -test). ouaino h rtclprmtr opoieats-e for test-bed controlled. multipurpose are a populations a provide different deliberate to these of for how parameters and allow critical generation whether likely the will optimal of model modulation the zebrafish The for compartment. organelle. organellar localisation, the a allows of modifies size for This overall However, which the usual by affecting 2010). change, without simply is achieved Marshall, shape as be PMEL-dependent can and compartment, functioning melanosome (Chan organellar differential this organelle of multicopy regulate to size number allowing the thus stage), developmental early this noteyl foe otocl-tg mro e xeiet with experiment, 5.5.2. Openlab per and microscope embryos dissecting imaged two-cell-stage Leica and examined a were to using larvae and one- embryos The microinjected of controls. was vehicle-only solution Danieau’s yolk of nl the 1.4 in into morpholino of ng 2 to Up gpr143 tgsaegvni asps-etlsto df t28 at (dpf) post-fertilisation days in given approximate The are 1994). (Westerfield, stages carried Book Zebrafish were the zebrafish to the according of out staging and collection maintenance, General injections embryo Zebrafish METHODS AND MATERIALS ertesato rnlto ag ra piejntos(p of (sp) junctions splice 5 at the or (atg) translation to of complementary start Tools, designed the near (Gene were Morpholinos OR) 1995). Philomath, al., LLC, et (Kimmel criteria morphological and ora fCl cec 21)18 4010 doi:10.1242/jcs.164400 1400–1407 128, (2015) Science Cell of Journal tyr eune,smaie nsplmnaymtra al S1. Table material supplementary in summarised sequences, 6 ... * s.e.m.; ˚ ,acrigto according C, pmela P 9 , sequence 0.05, , a(B), La pmelb ,

Journal of Cell Science net,B,Cree . izgn,A,Src,E . aai . opl,M., Coppola, S., Varani, M., E. Surace, A., Pizzigoni, K., Cortese, B., S. Incerti, M. Marks, J., A. Verkleij, S., Marco, T., and Boudier, R. J., W. C. Geerts, Hopkins, I., Q., Hurbain, A. Gomes, A., Rapak, M., L. Collinson, N., A. Hume, ib,D,Aain .M,Llo . iaoo . lm,A . te,K P., K. Steel, E., A. Klomp, Hellstro J., Kitamoto, C., Lillo, M., S. C. Azarian, M. D., Seabra, and Gibbs, W. M. Seeliger, B., G. Jaissle, S., J. Ramalho, E., C. Futter, B., Incerti, C., Valetti, C., Baschirotto, T., M. Bassi, A., Pizzigoni, M., d’Addio, F. W. Marshall, and H. Y. Chan, M. A. Laties, and B. Burnside, ismir . cremyr .adEb,O. Eibl, S. and M. U. Marks, Schraermeyer, A., and Biesemeier, G. Raposo, D., Tenza, C., D. Harper, F., J. Berson, he erfs o ahmrhln eeue o uniiaini each low- in using quantification least generated were for retina At full-length used of camera. images were Montaged morpholino device experiment. charge-coupled each for SC1000B imaged zebrafish and Orius TEM three 1010 Gatan Jeol a a on examined using and 200- cut and propylene were sections sections 70-nm serial and resin. nm epon series in embedded ethanol were zebrafish an the oxide, in dehydration Following ferrocyanide. potassium tetroxide-1% osmium paraformaldehyde-2% 1% 2% with incubation with fixed to prior were glutaraldehyde dpf) 5 and (2 embryos Zebrafish microscopy Electron ARTICLE RESEARCH as,M . cifio .V,Rnei . eNgi,F,Gli . rtii M., Bruttini, L., Galli, F., Nigris, De A., Renieri, V., M. Schiaffino, T., M. Bassi, C. King-Smith, and B. I. Barsoum, References at http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.164400/-/DC1 online available material Supplementary material Supplementary for PMC in Deposited M.S.]. and release.immediate and (D.F.C.) C.F. Council to 093445, Research number [grant Medical Trust Wellcome UK the the C.F.], to BB/D011841, number [grant Council Research Biological Sciences Biotechnology and the by funded was work This Funding authors all and manuscript. data, the the writing C.E.F. analysed to and and contributed M.N.O. work T.B., experimental project; the the performed conceived M.C.S. and D.F.C. C.E.F., contributions Author interests. financial or competing no declare authors The interests Competing data. microscopy electron model section to serial used the was 1996) from melanosomes al., et (Kremer IMOD length. by width by the calculated dividing was circularity and diameter, melanosome information the image measure to all used remove to was ImageJ used area. melanin of was measurement allowing melanosomes, excluding ImageJ in setting threshold subsequently,the and, Photoshop in images microscopy electron magnification e nihso h ahgnsso clrabns ye1. type albinism ocular of 2781-2788. pathogenesis the al. on et insights C. new D. Bennett, G., Jaissle, sheets. M., Seeliger, G., Jeffery, amyloid fibrillar of USA Sci. generation Acad. the Natl. and Proc. melanogenesis for G. implications Raposo, and melanocytes. in C. melanosomes M. Seabra, oiiyadlclzto fREmelanosomes. RPE of localization and S. D. motility Williams, and T. within R. distribution Libby, melanosome of regulation cells. the of epithelial in V. pigment cause Rab27a retinal M. of major role a Schiaffino, The (2004). is and OA1 A. of 1. Ballabio, processing type albinism and M., ocular transport Luca, intracellular De Defective M., Clementi, Press. University size. Harvard MA: Cambridge, 175-191. pp. Marmor), F. M. In and epithelium. pigment retinal the in mlatr eaooesaebthsol uteefc nvisible on effect subtle a only has but shape al. et melanosome J. pigmentation. Larsson, alters K., Wakamatsu, Pmel S., Ito, B., Ekesten, eaooe,lpfsi n eaoioucngaue fhmnREtissues. RPE Res. human Eye of Exp. granules melanolipofuscin and lipofuscin melanosomes, bodies. multivesicular Cell within Biol. morphogenesis Mol. premelanosome initiates Pmel17 eefroua liimtp rmtedsa hr r fteXchromosome. X the of arm short distal the from 1 Genet. A. type Nat. Ballabio, albinism and ocular A. R. for Lewis, gene A., A. 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