uui opsto ftehmnctpamcdni- complex -2 Asante* cytoplasmic David human the of composition Subunit ARTICLE RESEARCH 4774 2014 August 21 Accepted 2014; July 1 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 § UK. and 0RD, Science OX11 Harwell ` Harwell, MRC Campus, Unit, Innovation UK. Mammalian 1TD, Building, address: BS8 Sciences *Present Bristol Medical Bristol, Biochemistry, of of University School Laboratories, the of Biology Cell components with activated concert of transport in the acts drive to dynein-2 the complex which notably IFT-A flagella signaling, in as developmental pathway Retrograde well normal 1996). Hedgehog for al., as essential et cilia Criswell is 1994; IFT motile the al., and et (IFT), (Gibbons primary transport function which intraflagellar by of mediates process Vallee, complex component dynein-2 and retrograde and cytoplasmic the (Paschal the dynamics contrast, mitosis organelle By 1987). during traffic, these, of segregation membrane characterized in better involved the is being in dynein-1 exist minus-end-directed cells; complexes dynein primary mammalian cytoplasmic with the Two processes, motors. being these complexes. to and cytoplasmic crucial movement are the and accurate organelles motor the Microtubule intracellular on of depends positioning function cell Normal INTRODUCTION Intraflagellar Cilia, transport Dynein, motor, Microtubule WORDS: KEY identifying as or WDR34 well . either ciliopathy as candidate in disease, mutations cause why (also with WDR60 explain association LIS1 in data By found These dynactin, not dynein-1. are dynein-2. BICD2 regulators with and PAFAH1B1) does dynein-1 that as known it find common as also the dynein-2 We contrast, with respectively). associates DYNLL2, NudCD3 light and LC8-2 and and (DYNLL1 LC8-1 DYNLRB1) and chains as DYNLRB2), as known (also known (also (also TCTEX-3 roadblock-2 roadblock-1 and DYNLT3), DYNLT1) as as known known dynein-2, (also and function. dynein-1 TCTEX-1 cilia both for including to required common itself subunits several is define that We chain light as dynein-2 TCTEX1D2 the unique identify we a addition, time In function. We dynein-2 first for required complex. the dynein-2 ciliopathy the WDR60 for cytoplasmic by encoded cause proteins human define the to that show the known we of are intraflagellar Here, dynein-2 composition retrograde in ciliopathies. for mutations skeletal motor and the (IFT), transport is dynein-2 Cytoplasmic ABSTRACT hs uhr otiue qal oti work this to equally contributed authors These uhrfrcrepnec ([email protected]) correspondence for Author 04 ulse yTeCmayo ilgssLd|Junlo elSine(04 2,47–77doi:10.1242/jcs.159038 4774–4787 127, (2014) Science Cell of Journal | Ltd Biologists of Company The by Published 2014. r oafd yen2itreit hisadaeboth are and chains intermediate dynein-2 fide bona are , ` ioaL Stevenson L. Nicola , ` n ai .Stephens J. David and TCTEX1D2 WDR34 sa as and eedn ntetsu ye,tocpe foeo w light two of DYNC1I2, one or of copies (DYNC1I1 two type), chain tissue intermediate the on an depending of copies the by Schmidts (encoded 2013b; al., 2013; et al., Schmidts 2013a). et al., 2013; Huber et 2009; al., al., asphyxiating et and et McInerney-Leo III cause (Dagoneau among type complex dystrophy polydactyly 2011); dynein-2 thoracic rib the Beales, short of syndrome, and components Jeune (Waters in diseases human mutations ciliopathies of these, cohort the a to as lead Defects cilia known 2012). primary al., of et components function Liem key the and 2010; of in al., loading (Huangfu et the (Mukhopadhyay body Furthermore, for cilia required 2005). cell into be al., to the et known May to is 2005; IFT-A al., tip et cilia Liu 2005; the Anderson, from pathway this hi ce- ncnrligclalnt,peual in have presumably Others 2011). length, al., cilia et (Palmer controlling dynein-2 for in with light dynein association required the Tctex-1 for role is a defined chain WDR34 previously function and also that have cilia region We shown 2013). proper has pericentrosomal and work the ciliogenesis own Our to date. FAP133/ localizes the to 2007). of some of data al., explain context WDR34 the could et of which and outside (Rompolas complex, roles dynein-1 play flagellum DYNLL1 the to the the known as around is within localize LC8 known Furthermore, to and dynein-2 and (also was body motifs FAP133 LC8 algal basal binding 2013). humans) putative al., in of have DYNLL2 et present. Patel-King to chains 2007; also shown al., respectively) intermediate et are WDR60, (Rompolas and functional subunits WDR34 encode dynein-1 mammalian of known (orthologs other metazoans as Chlamydomonas the in such biochemical to composition organisms and motor Model subunit Tetrahymena identified, the lacking. been the of have is also of composition 2002)] [DYNC2LI1, al., subunit characterization chain et full intermediate (Grissom the light LIC3 (Criswell as and DHC2 known 1996)] and DHC1B al., as et known also [DYNC2H1, chain defined. well not complex, is dynein-2 2012). mammals, the al., in of particularly composition et molecular complex, (Splinter and the BICD2) dynactin contrast, PAFAH1B1) By (notably the as products are known gene al., these also bicaudal et (LIS1, of Splinter protein described 2009; lissencephaly-1 best Vale, been and The or one (Kardon 2012). have with partners associates and also binding dynein-1 more complex functions, its For dynein of molecules. majority cargo with the the association its for of controlling required in are implicated assembly chains families light correct Gull, The and 2009)]. the Tctex Wickstead Vale, 2005; and and Kardon al., 2007; et DYNLRB2) (Pfister DYNLT3) and (DYNLT1, (DYNLL1), chain (DYNLRB1 light dynein roadblock the of [including number subunits a chain and light DYNC1LI2) or (DYNC1LI1 chains intermediate yen1i opie fadmro ev hi subunits chain heavy of dimer a of comprised is Dynein-1 lhuhseii ee noigadni--pcfcheavy dynein-2-specific a encoding genes specific Although § aepoie ute le htequivalents that clues further provided have DYNC1H1 ee noigFP3 n FAP163 and FAP133 encoding genes ee,wihascaewt two with associate which gene), nvitro in Chlamydomonas Aat tal., et (Asante and

Journal of Cell Science oc-uiywt A13adL8 n,i h planarian the in and, LC8, and mediterranea FAP133 with Schmidtea co-purify function. to dynein-2 in WDR34 for WDR34– role consistent Furthermore a LC8, 2013a). with FLAG-tagged is al., with (WDR34–tGFP) et co-immunoprecipitates WDR34 (Schmidts tGFP of cilia fusion in protein present and fluorescent region pericentrosomal a a to that localizes WDR34 that shown also ARTICLE RESEARCH nw sTtx2.Ti uui sntfudi soito with association (also in found TCTEX1D2 not is detected subunit This we and Tctex-2). as addition, Tctex-3, known with and association In light in Tctex-1 identified dynein-1 mGFP–WDR34. were LC8-2, known roadblock-2) and and all roadblock-1 (LC8-1 Here, chain identified. isoforms we dynein chain heavy the that DYNC2LI1/ are the chains S1B dynein-2 subunit Table light abundance) material chain known supplementary low in intermediate Listed at the LIC3. light (albeit as dynein-2 detected known well also seen We as also (DYNC2H1). were spectra WDR60 and this peptides for using of mGFP–WDR34 numbers of Large isolation approach. 2006). S1A efficient Table highly material al., the were Supplementary shows that experiment. components et this dynein-2 for in putative (Rothbauer data identified and dynein-1 the GFP-nanotrap known proteins shows all S1 a other Table material the using proteomic Supplementary define a that mGFP with took to mGFP–WDR34 we of of line this, interactome the achieve cell comparing To approach this WDR34. use with associated to sought the We mGFP– of component a of complex be dynein-2 cilia. to the in localization pericentrosomal mGFP–WDR34 localized reveals not of Proteomics the is to alone GFP that that as specific, localizes is from shows cilia to WDR34 1F differs which Fig. cilia structures; protein, ( within localization body the endogenous that the mGFP–WDR34 basal shows 1E of the Fig. of base 1B–D). of Fig. the OFD1, components and acetylated at ODF2 co- known colocalizes by against with also cilium confirmed directed mGFP–WDR34 cytoplasmic was 1A). antibodies diffuse (Fig. cilia a with to showing in labeling localization as that cilia This shows well primary distribution. 1 as and Fig. cells (mGFP). centrosomes GFP serum-starved WDR34 to of localizes expressing form monomeric mGFP–WDR34 stably a line to fused cell pigment (hTERT-RPE1) retinal telomerase-immortalized epithelial human a generated We cilia to localizes mGFP–WDR34 RESULTS motor. dynein cytoplasmic determine second of function to characterized the poorly subunit and modulate this the also complex define regulators dynein-2 dynein-1 to dyneins known out human whether set complex axonemal the we dynein-2 of detail, the metazoan in composition described a and been Because not 2000) 2007). for has al., cytoplasmic al., LIS1, both et with et (Pedersen complexes. concert (Faulkner in accessory function dynein-1 to dynein-1 known also is other with example, It cooperation or in lacking. functions are dynactin dynein-2 this whether human unclear validating of remains components data with 2013; key consistent biochemical skeletal being al., are dynein-2, WDR60 data et possibly cause these and of McInerney-Leo Although WDR34 2013a). (WDR60) 2013; orthologs al., al., human et FAP163 Schmidts et the (Huber and in ciliopathies mutations (WDR34) several that from FAP133 shown work Recent has 2013). groups al., et (Patel-King ciliogenesis A13hsas ensont oaiet h lgla matrix, flagellar the to localize to shown been also has FAP163 ob boueyrqie for required absolutely be to , c -tubulin, sdteepoemcdt ogieorfrhrbiochemical further our guide known to are then data which We all. of proteomic analyses. at detected all these not NDEL1), were used dynein-1, as NDE1) for known as factors known accessory (also (also NudE Nudel of that Spindly, and above 2, levels our Bicaudal not at in LIS1 alone. does or mGFP detected 1 mGFP–WDR34 or bicaudal were that dynactin, that with NudC-like data show associate factors These the called accessory analysis. shows dynein proteomics (also S1D key the NudCD3 Table for material and Supplementary NudC NUDCL). dynein- of function, regulators two 1 with S1C) mGFP–WDR34 Table of material association (supplementary the noted also We alone. mGFP p50 D6 hwdta hswshgl fiinl sltdin isolated efficiently two these for highly that implying Immunoblotting was 2B), (Fig. this protein. mGFP–WDR34 with that complex endogenous a form showed the not WDR60 does the with form recombinant that complex the mGFP– that found with suggesting co-immunoprecipitate we WDR34, not did Importantly, are WDR34 specific mGFP–WDR34. endogenous the WDR60 shows of 2A and Fig. isolation WDR34 immunoblotting. were using findings both validated These complex. then that dynein-2 the indicate of chains intermediate data above human The the of interactions complex and dynein-2 components the of Validation a eaiecnrlpoen AD Fg D.Two 2D). (Fig. GAPDH p150 protein, complex, dynactin control the negative of components nor DYNC1I2), a 2C), same (Fig. as canonical mGFP–WDR34 the was known the with (also immunoprecipitated in data, not IC74 proteomic was present chain, the intermediate be from dynein-1 expected might As chains complex. intermediate putative AD skont soit ihmn te proteins other many the explains with likely expected. which associate 2013), as al., to et fractions (Zala dynein-1 known was including low-density GAPDH, is protein in control mGFP GAPDH the contrast, predominantly of By majority found the complex. like dynein-2 3B), intermediate same (Fig. as the WDR34 role within their large with that chains in consistent As found 3B,C), complex. both (Fig. suggests were high-molecular-mass complexes WDR60 a dynein-1 and This of mGFP–WDR34 the context expected, 3A). to the (Fig. in fractions functions similar chain in intermediate on gradients sedimented that density we cytoplasmic complex complex, large sucrose a to macromolecular in large WDR34 similar endogenous a found for coefficient, predicted sedimentation As dynein-1. high a with available of lack been a have to intermediate We owing light antibodies. immunoblotting, or shown). by chain not heavy subunits from dynein-2 (data chain downs the cells analyze pull to RPE-1 dynein-1 unable and these in HeLa BICD2 identified both and always LIS1 five dynactin, also in dynein-1; were determined of as downs but dynein-1, pull NudCD3, independent with and associate Tctex-1 the TCTEX1D2, data both not These but because 2J). specificity, (Fig. lysates some identify immunoprecipitates 2J). reveal in whole-cell (Fig. detection to mGFP–WDR34 its in of enables mGFP–WDR34 able isolation detectable of GFP-nanotrap following with were not enrichment identification we is association By our TCTEX1D2 proteomics, both in were 2G). with our (Fig. TCTEX1D2 2I) (Fig. in Consistent detected NudCD3 TCTEX1D2 isolated. not and mGFP– 2H) was efficiently (Fig. with Tctex-1 LIS1 immunoprecipitate contrast, to similarly, failed WDR34; also respectively) yen2i rdce ob ag-oeua-ascomplex large-molecular-mass a be to predicted is Dynein-2 dynamitin ora fCl cec 21)17 7448 doi:10.1242/jcs.159038 4774–4787 127, (2014) Science Cell of Journal Fg F as nw sDT1adDCTN2, and DCTN1 as known (also 2F) (Fig. Glued Fg E and 2E) (Fig. 4775

Journal of Cell Science EERHARTICLE RESEARCH swl ssm GPisl Fg C,ms ieyaiiga a as arising complex likely most the 4776 3C), (Fig. across itself mGFP observed, WDR60 some was gradient as of mGFP–WDR34 well unincorporated as distribution higher-density Some significant 3C). the no the (Fig. had on mGFP–WDR34 across of effect expression detected The fractions. amounts low paetmlclrms ftemGFP–WDR34-containing the of the mass determine to Further lysates molecular 1. cell of Fig. filtration in apparent gel seen explain undertook labeling we also this, cytoplasmic could to diffuse This the WDR34. the of and at much protein mGFP recombinant between the site of linker processing proteolytic of result ora fCl cec 21)17 7448 doi:10.1242/jcs.159038 4774–4787 127, (2014) Science Cell of Journal eosrt nasneo F rmpiaycla Scale cilia. primary to from 5 E GFP bars: in of as absence processed an were demonstrate shown. GFP are expressing examples Cells Two (F) indicated. tubulin as acetylated WDR34, detect and to with labeled fixed and images. were paraformaldehyde main mGFP–WDR34 the expressing in Cells indicated (E) show areas Insets the length. of its enlargements along with cilium evident the the labeling of demonstrate some base red) the (all at mGFP–WDR34 OFD1 of (D) accumulation (B) and of ODF2 cilia, Labeling (C) primary (red). tubulin, to tubulin and acetylated pool with cytosolic colocalizing a to localizes mGFP–WDR34. (green) of Localization 1. Fig. m m. A mGFP–WDR34 (A) c -

Journal of Cell Science EERHARTICLE RESEARCH muolte odtc A D3,()WR0 C C4(DYNC1I2), IC74 (C) p150 WDR60, (E) (B) GAPDH, and WDR34, (D) SDS-PAGE (A) by detect separated to were immunoblotted mGFP–WDR34 or alone stably mGFP mGFP–WDR34. cells or RPE1 mGFP either serum-starved expressing from Immunoprecipitation 2. Fig. rcin(U)adtebudfato ‘’,a niae.Teatrs nJ in asterisk The unbound indicated. the as (‘I’), (‘B’), bands. input fraction non-specific the bound indicates show the lanes and are the (‘U’) markers case, fraction Molecular-mass each indicated. In as (kDa). TCTEX1D2, shown (J) or NudCD3 (I) Glued F p50 (F) , dynamitin G I1 H Tctex-1, (H) LIS1, (G) , F-rp fclsexpressing cells of GFP-traps D3 hti otie ihntedni- complex. dynein-2 mGFP– the of within proportion contained exact is the that of WDR34 analysis fractions. the low-density complicates and This low-molecular-mass fraction from in product significant elutes degradation a that a as is this) mGFP there also that (and mGFP–WDR34 evident of is it gradients, sucrose lolclzdaon h etooe(i.4)adta this that and dynein-2 4A) WDR34, (Fig. same like centrosome WDR60, the the endogenous around within that that localized act suggest also found above might We shown WDR60 complex. data and biochemical WDR34 role of The the true examined WDR60. was first this we of whether subunits, dynein-2 determine lengthening identified To a newly remain. also the that but cilia cilia a produce those in that of results cells proteins of these 2013) number of reduced al., either of et and depletion (Asante formation siRNA-mediated al., WDR34 cilia et (Palmer and in Tctex-1 of 2011) defects depletion following that evident are previously function function shown cilia have and ciliogenesis We for required is WDR60 column mGFP–WDR34 the of detectable high-molecular- to volume corresponding all ml) void ( 7.9 components mass 15, the that fraction 6 with 3D, Superose (Fig. found calibrated eluted a fluorescence we using that, shows column, 3D Fig. complexes. hrceie rvosy(snee l,21) ihsRA#1 for siRNA with previously been 2013), seen have al., have giantin et target area we (Asante to pericentrosomal previously used as characterized duplexes the 5D) siRNA the in The in with WDR34. WDR60 correlated quantified also of giantin 5C, (Fig. labeling of loss in of (quantified of centrosome intensity degree the The at giantin 5B). labeling in of Fig. WDR60 same reduction loss a of the that in amount result shows the predict indeed are 5A does would Fig. WDR60 depletion siRNA-mediated WDR60. one and by for then true WDR34 hold together If would complex 2013). as in al., known indeed (also et of giantin function (Asante protein the GOLGB1) matrix on Golgi dependent WDR34 transmembrane was of the region localization pericentrosomal the that the showed to we work, previous dependent our is In cilia WDR34, of the that giantin like on WDR60, exacerbate of localization further The not 4H–J). did (Fig. both seen both WDR60 of phenotypes using depletion and analysis double note, our for Of WDR34 repeated required siRNAs. then WDR60 reciprocally We and revealing WDR34 each other. as the were well of WDR60 as stability 4G), and (Fig. WDR34 siRNAs that WDR60 efficacy we the and confirmed what WDR34 Immunoblotting cumulative of 2013). a to 4F). on al., (Fig. evident the similar plot particularly et is frequency of length highly (Asante cilia length WDR34 in is increase in of The this increase depletion of following clear 4E); loss of (Fig. observed a proportion that with cilia the 4D), in showed remaining (Fig. reduction data cells significant a ciliated these WDR60 in of #2), of resulted labeling siRNA of WDR60 Quantification loss WDR60 a for in 4C). with two associated (Fig. 4C shown were of extended Fig. which an (example of of in one loss both length complete shown to a (example or cilia #1) led axoneme siRNA in centrosome WDR60 clearly for increase the 4C Fig. around WDR60 an WDR60 labeling – of or WDR34 phenotypes Depletion WDR34 of either 4B). loss (Fig. of together. a function in depletion they resulted this, that suggesting with itself 4A), Consistent WDR60 (Fig. either WDR34 of of depletion or following lost was localization h ag f4–7 of range the ora fCl cec 21)17 7448 doi:10.1242/jcs.159038 4774–4787 127, (2014) Science Cell of Journal 6 10 , 4 1 6 a(i.3,fatos3–7.A ihthe with As 33–37). fractions 3D, (Fig. Da 10 7 a.GP ycnrs,eue within eluted contrast, by GFP, Da). nvitro in Specifically, . 4777

Journal of Cell Science EERHARTICLE RESEARCH elne Fg D uniidi Ewt uuaiefrequency 6C). cumulative in with 4778 to 6E found quantified in were quantified 6B, remain 6D, (Fig. to of (Fig. seen ability longer cilia were in be the that primary NudCD3 diminished cilia those this of generate using Furthermore, that role NudCD3 to found the of and cells test expression 6A) (Fig. to the siRNA suppressed assays We similar ciliogenesis. used WDR34 of then localization the We for required is NudCD3 the 5D. from evident Fig. is in also seen This #2. data siRNA than effective more being fetn h oaiaino F1(i.6)o pericentrin the or around 6I) (Fig. WDR34 OFD1 of of without loss 6B). localization 6H) (Fig. Fig. a the in quantified of in affecting 6G; depletion (Fig. data, resulted region other pericentrosomal our also with Consistent NudCD3 6F). in shown plots rnfcin i.7 hw h fiayo he individual siRNA three using in shown of As TCTEX1D2 (QPCR). efficacy PCR of quantitative the by role measured shows as siRNAs, the 7A Fig. examined transfection. function then cilia normal We for required is TCTEX1D2 ora fCl cec 21)17 7448 doi:10.1242/jcs.159038 4774–4787 127, (2014) Science Cell of Journal rp,aindt h rcinnumbers. fraction the the to below aligned shown graph, is calibration mass Molecular bars). and (black bars) cells of (gray mGFP–WDR34-expressing filtration cells Gel GFP-expressing (D) from lysate. lysates cell whole L, and WDR60. GAPDH DIC74, GFP, and for SDS-PAGE immunoblotting by analyzed were fractions and alternate 500- post-centrifugation 20 collected of were total fractions a A onto gradient. density loaded sucrose and 5–20% lysed GFP were (B) mGFP–WDR34 expressing (C) stably or cells or RPE1 WDR34 (B) (DYNC1I2), GAPDH. DIC74 of across gradient distribution the the by determine probed to and immunoblotting gradient the were from fractions removed density 1-ml sucrose centrifugation, 5–40% After a gradient. onto loaded and cells. lysed RPE1 from of lysates centrifugation gradient cell density Sucrose 3. Fig. A P1clswere cells RPE1 (A) m l

Journal of Cell Science EERHARTICLE RESEARCH i.4. Fig. e etpg o legend. for page next See ora fCl cec 21)17 7448 doi:10.1242/jcs.159038 4774–4787 127, (2014) Science Cell of Journal 4779

Journal of Cell Science eietooa D6 aeigcreae ihteefcc fgatnsprsin nAC h ra ulndaesona ihrmgiiainbe tra magnification siRNA higher indicates at coding Color shown coefficients. are Spearman outlined and Pearson’s areas both the using 10 tested A,C, bars: was In correlation Scale suppression. the indicated. and giantin as quantified of were efficacy Data (D) the image. with main correlates labeling WDR60 pericentrosomal 10 bars: Scale I. 0.25- percentage in the in shown of found chart cilia frequency total cumulative of a as represented length cilium ii nrsos o2- eu train E h egh fteremaining the of lengths The (E) produced starvation. that serum cells 24-h in of to percentage indicated response the areas in of (example the cilia Quantification cilia of (D) generate enlargements images. to show main failure Insets the a #2). or WDR60 #1) for WDR60 shown for longer of shown either loss (example showed by labeling cilia (shown and WDR60 centrosome), of the or depleted around WDR60 effectively immunoreactivity were (A) and Cells and (C) fixed (AcT) WDR34. were tubulin (B) cells acetylated starvation, detect or serum to WDR34 following immunolabeled targeting and duplexes indicated, interdependent. siRNA as are with WDR60, WDR60 transfected and were WDR34 Cells of (A,B) stabilities The 4. Fig. ARTICLE RESEARCH 4780 mean the show Data plotted. and measured was WDR60. labeling pericentrosomal WDR60 of pericentrosomal loss of a area in The results (B) giantin WDR60. of Suppression 5. Fig. in of ability evident the more Notably, 7D). is (Fig. length cilia This plots in frequency depletion. increase TCTEX1D2 cumulative cases, significant of TCTEX1D2 all statistically In depletion a following withdrawal. detect serum 7C, can Fig. following we cilia in longer in quantified results and 7B Fig. D,E,H,I in Data of seen. length was the effect (I) additive mean or no the cilia cases show producing both cells In of cilia. in percentage remaining seen the both phenotypes (H) the of of enhance depletion terms could whether simultaneously determine WDR60 to and indicated analyzed WDR34 are were negative markers Results Molecular-mass a (H,I) control. as (kDa). loading the included a test is as to A/C GAPDH used Lamin control, also subunits. was these versa of vice interdependency of following and efficacy WDR34 WDR60 the for of confirmed Immunoblotting suppression Immunoblotting siRNAs. (G) WDR60 E. and in WDR34 shown 0.25- that chart in as found frequency same cilia cumulative total a of as cultures. percentage represented the cell of length WDR60-depleted cilium or of (GL2) Distribution control (F) for measured were cilia 6 .. * s.d.; P , m .5 ** 0.05; m is h aapotdaetesm sthat as same the are plotted data The bins. m m. m m. P , .1 *** 0.01; m is h aapotdaethe are plotted data The bins. m P , .0.()Dsrbto of Distribution (J) 0.001. A el eltdo ini eelbldt eetaeyae uui AT and (AcT) tubulin acetylated detect to labeled were giantin of depleted Cells (A) el ofr rmr ii sntovosyafce following affected obviously not is 7E). (Fig. cilia depletion primary TCTEX1D2 form to cells rvd oefntoa pcaiainadgetrcnrlof control greater and and specialization WDR34 functional to both potential more the dynein-1 contains has Our provide asymmetry dynein-2 dynein-2 the chain. This fact, chains. which intermediate intermediate to in WDR60 the in that, analogous of model suggest copies FAP133, data a two of propose containing copies complex to two authors and contains the 2007), a al., led being et FAP133 (Rompolas they with subunit consistent dynein chain are intermediate the data dynein-2 with These and LC8. subunits chain, chain) light (heavy intermediate light dynein-2-specific and other using chain most with experiments in co-immunoprecipitates Biochemical and DYNC1I2 neurons). (typically in Chlamydomonas chain DYNC1I1 intermediate and cells, chain dynein-1 intermediate light same cytoplasmic the same of WDR60 copies data Individual and two Our contain WDR34 subunits. complexes 8. both Fig. chain contains in dynein-2 schematic intermediate of human the composition that in subunit the summarized show the to as of of reports motor, previous picture characterization this on comprehensive molecular build a data first provide Our complex. the dynein-2 provide human we Here, DISCUSSION ihrWR4o h Csbnt fdni-.I stmtn to tempting Evidence, function. is its It from to than dynein-1. related primarily is of protein asymmetry subunits this larger IC that a speculate the or is WDR34 WDR60 either Furthermore, function. motor yen oti utpeitreit hiswti h same the within chains intermediate multiple contain dyneins ora fCl cec 21)17 7448 doi:10.1242/jcs.159038 4774–4787 127, (2014) Science Cell of Journal Chlamydomonas aesonta A13(D3)co-purifies (WDR34) FAP133 that shown have 6 .. *** s.d.; P , .0.a,abtayuis C h osof loss The (C) units. arbitrary au, 0.001. eosrtsta axonemal that demonstrates , nsfection o the low

Journal of Cell Science EERHARTICLE RESEARCH i.6. Fig. e etpg o legend. for page next See ora fCl cec 21)17 7448 doi:10.1242/jcs.159038 4774–4787 127, (2014) Science Cell of Journal 4781

Journal of Cell Science uui,gen.Arw niaeeape fclaeegn from emerging 10 cilia bars: (acetylated of Scale centrosome examples centrosomes. the indicate around Arrows (red) green). OFD1 tubulin, of accumulation the affect vdn.()Qatfcto hw hti el eltdo uC3that NudCD3 of depleted cells in are that cilia shows which in Quantification quantified. those (E) show was cells evident. in cells NudCD3-depleted body ciliated of basal examples of Further the proportion (D) from The cilia (C) extend cells. to centrosomes. NudCD3-depleted failure from emerging the cilia highlight and acetylated of Enlargements control examples and indicate pericentrin siRNA Arrows detect an (AcT). to as tubulin immunolabeled A/C were lamin Cells control, (B) loading NudCD3. a Immunoblots as (A) control. tubulin used. length show were cilia NudCD3 and targeting ciliogenesis duplexes for siRNA required independent is NudCD3 6. Fig. ARTICLE RESEARCH r ossetwt u oe htbt c oehri the dynein-2 in full together the versa act 4782 that vice both conclude and that We WDR60 model complex. of our dynein-2 function with same cannot of consistent WDR34 seen loss that are findings those for The 2013b). to al., compensate al., et (Dagoneau phenotype et chain Schmidts heavy similar 2009; dynein-2 the a of mutation causes with following 2007) 2013; al., ciliopathies et al., is (Rompolas et WDR60 skeletal (Huber or function 2013a) WDR34 al., either et dynein-2 of Schmidts without Mutation (Wickstead 2007). for evolution Gull, chain through and of conservation subunits their importance heavy by chain the underscored dynein-2 However, intermediate WDR60. functional chain these and heavy the WDR34 out a the rule chain associated not of do of intermediate therefore loss also the data concomitant possibility These of 2011). in al., loss result et (Palmer that not did shown work subunit has previous monitor our to dynein-1 immunoblotting, able by with been chain not heavy also have dynein-2 are we the experiments Although filtration WDR60 this. gel with with Our complex consistent complex. that heterodimeric other complex a some dynein-2 in in the or isolation, of in outside act with WDR34 could association of in there proportion being that a shows WDR60 co-immunoprecipitation is centrifugation all gradient our of density Sucrose of favor WDR34. in basis dynein-2 argue the we assembled data, high-molecular- On presence with the the complexes. association with in of mass of or consistent within pool are some context mGFP–WDR34 data has a of filtration is itself the gel exposing WDR34 dynein-2 our of that cilia, complex; that out outside primary or rule base cannot of function also cilia base We the the WDR34. at IFT-A at factors different the assembled a associated as indicate of such could factors, set labeling is the pericentrosomal other to epitope The binding to antibody dynein particle. or to the due that be Schmidts could axonemal suggest 2013; This al., data cilia. et within These Asante occluded 2013a). antibodies also (see al., available pool et currently this detect with not labeling does cilia, in these detectable of association to the similarities microtubules. to axonemal shows relate with dynein-2 could motors This cytoplasmic dyneins. such, axonemal As IC2. from and dynein arm chain outer intermediate Dimeric Chlamydomomas IC97. three and contains IC138 I1 For IC140, 1991). subunits dynein al., arm et inner King 1986; example, Rosenbaum, and (Mitchell complex horizontal the by shown is mean the and * acetylated quantified line; by with were of shown labeled Data Depletion as are (H) (G) (red) cilia tubulin. E. WDR34 and in pericentrosomal centrosome shown of The that loss immunofluorescence. 0.25- as a in same in found the results cilia are NudCD3 total plotted of data percentage The in the bins. Data of cells. chart control frequency in cumulative seen mean those the than show longer C,E were these cilia, extended ti neetn ont htatog GPWR4is mGFP–WDR34 although that note to interesting is It P , .5 ** 0.05; P , .5 *** 0.05; 6 otistoitreit hi uuisIC1 subunits chain intermediate two contains ..()Dsrbto fclu eghrpeetda a as represented length cilium of Distribution (F) s.d. P m , m. .1 I upeso fNdD osnot does NudCD3 of Suppression (I) 0.01. Two m m elto xeiet n o opeekoku.The knockout. only complete are a experiments not these and as of formation, experiments is depletion TCTEX1D2 cilia depletion that TCTEX1D2-depleted idea following for the note, reject cannot observed required length Of We cilia. we form cilia giantin. still cells and in which in WDR60 increase reduction that WDR34, an a to that in show similar results data cargo-binding expression Our the complex. TCTEX1D2 influence the potentially of to capabilities and of binds regulation function the TCTEX1D2 similar diversify could dynein-2 functionally of This a WDR60. copy (or to chain binding single 2001). protein) light alternative al., a an et that with WDR34, (DiBella is monomeric dynein possibility other is One unlike TCTEX1D2 that, suggests chains, Evidence dynein light 1999). arm al., outer et dynein-1. control Pazour in to present in found chain not been assembly light dynein-2, previously dynein-2 TCTEX1D2 for has chain the with Tctex-2 light identify a al., also Tctex-1 as et now (Tctex-2) (Palmer of we work previous Here, association our 2011). from The predicted was cilia. dynein-2 complexes which from) retrograde in (and within way exit distribution the protein controls to and clues assembles important provides biology IFT in operates WDR60, and cilia. WDR34 within both including complex, fclad o elc h situation also the is reflect not It directing do binding. cilia in cargo our of act in that note chains and/or to important assembly light complex dynein It dynein whether system. this in unclear chains light remains dynein other with overlap here functional seen phenotype moderate rmptet ihWR4mttosso hre ii on cilia shorter show mutations cells WDR34 indeed, with have mutations; cilia patient longer patients for that extension from note described no to been or important limited typically also with is not cilia, It form axoneme. to the results cells of axonemal depletion of effective deliver inability More an to tip). in the continues to IFT components other anterograde and in results (as our and cilia IFT of We retrograde longer compromises effect longer. function were threshold of loss remained a Partial that as those – this but subunits interpret cilia fewer dynein-2 were of there suppression siRNA-mediated following 2007). al., localize et to (Pedersen shown cilia been dynein-2 has motile or NudC we to cilia that in yet, note NudC we of of proteomics however, role as function; our the but, further in investigated S1C) not protein have Table NudC material related (supplementary the analysis loss-of- identified death. a cell also to as leading We ciliopathies, likely dynein-1, with compromise find associated would to function its gene expect to this not linked in would likely we mutations most However, is dynein-2. ciliogenesis with function in interaction both its role with of Its NudCD3 analysis within complex. of the more dynein-2 association makes of The complexes assembly cell. dynein the the cytoplasmic of in suggests body role which main a 2006), the plays or al., protein immunofluorescence et this by (Zhou that cilia forms not in it have epitope-tagged NudCD3 We using and detect dynein-2. to with 2006), role able al., consistent similar been are a data et playing Our NudCD3 2009). (Zhou al., with et complex (Cai centrosomes dynein-1 to localizes the experiments of siRNA the stability our of IFT. formation that retrograde the in is defect i.e. a interpretation ciliogenesis, reflect in Our be axoneme. to appears failure nlsso yenlgtcanfnto ntecneto cilia of context the in function chain light dynein of Analysis noreprmnsw yial bevdtooutcomes two observed typically we experiments our In and assembly the influence to shown been has NudCD3 ora fCl cec 21)17 7448 doi:10.1242/jcs.159038 4774–4787 127, (2014) Science Cell of Journal Chlamydomonas nvitro in lgla(ae-ige l,1997; al., et (Patel-King flagella nvitro in sasrpriga elongation an reporting assays nvivo in ih lorfetsome reflect also might nvitro in hr h primary the where experiments.

Journal of Cell Science el on ncnrladTTXD-eltdcls hr sn ossetcag olwn iN ramn.Dt hwtemean the show Data treatment. siRNA following change consistent no is There cells. TCTEX1D2-depleted 0.25- and in control found cilia in total found of cells percentage the of chart frequency cumulative oprn ii egh ncnrladTTXD-eltdcls aaso h mean the show Data cells. TCTEX1D2-depleted and control in lengths cilia comparing EERHARTICLE RESEARCH o eanascae ihi uigognletasot(Egan transport organelle during it does but with motility associated dynein-1 of remain not For initiation dynein-2. the not do with in data transiently acts our associate LIS1 dynein-2, not example, with do associate they dynactin that stably that preclude not show does do work LIS1 our and although that however, is, note It to LIS1. important and dynein-1 dynactin cytoplasmic including dynein, of axonemal regulators and known dynein-2 many with the dynein-2 of of WDR34, for context roles the clear within show complex. TCTEX1D2 data and our WDR60 Nonetheless, data. between the distinction This can, 2013). and mutations others WDR60 al., while with et patients axoneme from (McInerney-Leo an fibroblasts variability, in extend seen phenotypic to been has failing However, cells 2013). some al., with et (Huber average 10 bars: Relat Scale (A) depletion. pericentrin. TCTEX1D2 and upon (AcT) length tubulin cilia acetylated in detect increase to the immunolabeling highlight and Enlargements fixation mean cells. the (B–D) depleted show or Data and RT-PCR. QPCR control by by determined of as analysis projections cells, (A) transfected to in expression prior mRNA h TCTEX1D2 24 length. for cilia starved increases serum depletion TCTEX1D2 7. Fig. ti losgiiatt oeta ed o eetayassociation any detect not do we that note to significant also is It nvivo in ucmsncsiae ato nteitrrtto of interpretation the in caution necessitates outcomes P1clswr eltdo CE12uigtredfeetsRAdpee sidctdadthen and indicated as duplexes siRNA different three using TCTEX1D2 of depleted were cells RPE1 nvitro in m is h aapotdaetesm sta hw nC E h ecnaeo ciliated of percentage The (E) C. in shown that as same the are plotted data The bins. m yen2i ikn h oo oiscro te recent with Other as-yet- cargo. cooperation other its in to that motor act the suggests linking likely al., in dynactin et factors dynein-2 Moon of 2013; accessory al., absence et unidentified Wang The (Egan 2012; al., motility 2014). et subsequent Splinter and 2012; the cargo al., direct et with to dynein cooperate likely of LIS1, proteins dynein-2. association family by transported BICD nuclear repertoire and cargo loads dynactin the in cargo of large part such example not that cilia conceivable are of is (for force it the beating), flagellar dynein-1 generating and (in dyneins of axonemal and required migration) co- such Although are 2013). by in al., function functions et its (McInerney-Leo aid dynein-2 to loads we dynein heavy of moving with by state although force act persistent cilia to a association believed generating primary model, also in is to LIS1 in a shown). LIS1 not (data localize immunofluorescence LIS1 such can with detect we immunoprecipitation, Consistent not 2012). do al., et 6 s.d.; ora fCl cec 21)17 7448 doi:10.1242/jcs.159038 4774–4787 127, (2014) Science Cell of Journal n 5 ;*** 3; 6 s.e.m.; P , .0.()Dsrbto fclu eghrpeetda a as represented length cilium of Distribution (D) 0.001. n 5 .()Rpeettv aiu intensity maximum Representative (B) 3. 6 s.e.m. m .()Dtplot Dot (C) m. nvitro in z -stack 4783 ive

Journal of Cell Science EERHARTICLE RESEARCH im-lrc) abtplcoa nigatn(R-1C,mouse (PRB-114C), 4784 anti-giantin polyclonal rabbit anti- monoclonal Sigma-Aldrich); rabbit mouse and anti-acetylated anti-WDR60 (HPA019136), (T6793) monoclonal tubulin polyclonal mouse anti-NudCD3 (HPA001874), rabbit anti-ODF2 polyclonal include: polyclonal study rabbit this (HPA020607), in used Antibodies Antibodies by serum of 37 rid at incubated were were and cells PBS with hTERT-RPE1 twice confluence, washing reaching On assay Ciliogenesis 35-mm L- MA). onto Ashland, 1% seeded (MatTek, were with cells dishes Cells with supplemented amino-acids. glass-bottomed essential UK) supplemented 1% epithelial Paisley, and DMEM-F12 Technologies, glutamine (Life pigment in FCS maintained 10% retinal were (hTERT-RPE1) telomerase-immortalized Human cells culture unless of Growth UK) (Poole, Sigma-Aldrich from stated. purchased otherwise were disease. reagents All cause candidate METHODS AND a MATERIALS gene as either TCTEX1D2 identifies in gene. ciliopathy work mutations this a function, Furthermore, dynein-2 defines why in dynein-2 WDR60 and cytoplasmic explaining WDR34 of both composition for requirement subunit characterization our the conclusion, In of questions. such undoubtedly address would to are analysis help biophysical factors and itself. additional complex biochemical processive that IFT-A Further 2014; the speculate a possibly processivity, be to al., induce to tempting to et required likely is also (McKenney it is and state Dynein-2 motor, 2014). processive al., a et as Schlager such induce factor accessory to another BICD2 and dynactin the both requires dynein-1 of association that shown have experiments reconstitution sn h iiipeetto fIaeJ(cidlne l,2012). anti- al., measured et with (Schindelin were J lengths labeled Image Cilia of typically cilia. implementation Fiji and primary the subsequent mark fixed using and to exit were tubulin cycle Cells acetylated cell induce assembly. to medium cilium serum-free in h 24–48 c tbln(65)(l from (all (T6557) -tubulin ˚ ne %CO 5% under C 2 for E23 el sn h Lenti-X the using cells HEK293T secondary by facilitate analysis. validated Kit alignment to sequence were Miniprep sequenced by minipreps subsequently Spin validation and plasmid QIAprep analysis All using restriction UK). cells Manchester, bacterial and (Qiagen, the (Stratagene) cells was from by bacterial cDNA harvested ultracompetent flanked pLVX-Puro-mGFP-WDR34 XL10-Gold sequence in resultant amplified mGFP The sites. the restriction includes View, Mountain that (Clontech, vector CA) pLVX-Puro modified a into subcloned Xho GAAAGAATTCTCAGGCCGCCACCTCTGCTGC-3 n Ry 8 okyat-abtIG(2-22)wr rmLi-Cor from were (926-32223) anti-rabbit-IgG UK). (926-32212) donkey (Cambridge, anti-mouse-IgG Scientific, 680 donkey Stratech IRDye 800CW (from and IRDye ImmunoResearch UK). Jackson (711-035-150)Newmarket, by anti-rabbit-IgG supplied donkey were peroxidase-conjugated and 150) (715-035- anti-mouse-IgG anti-rabbit-IgG donkey peroxidase-conjugated donkey donkey (711-165-152), Cy2-conjugated Cy3-conjugated UK). (715-225-151), Leicester, Cambridge, anti-mouse-IgG Laboratories, of Bioscience, Bethyl University (A300-409A, Cambridge anti-LIS1 Fry, from polyclonal Andrew rabbit from and gift UK); polyclonal generous rabbit UK); (a Hitchin, anti-OFD1 Technologies, Signaling Cell (2032, anti-Lamin A/C polyclonal rabbit UK); anti- Feltham, monoclonal Millipore, (MAB1618, mouse DIC74 UK); Cambridge, Abcam, mouse from (both TX); (ab139804) anti-TCTEX1D2 polyclonal Dallas, rabbit anti-WDR34 polyclonal and Biotechnology, (ab9484) anti-GAPDH polyclonal rabbit Cruz monoclonal Santa UK); rabbit (sc-28537, Cambridge, UK); anti-Tctex1 Biologicals, Oxford, Novus Biosciences, (NBP1-88805, (both 611003) BD (BD anti-p50-dynamitin from monoclonal mouse anti-p150-glued monoclonal and mouse (610473) CA); Covance, from anti-pericentrin (all polyclonal (PRB-432C) rabbit and (MMS-118P) anti-GFP monoclonal iLb,Hthn K ihtefradpie 5 primer forward the with UK) TCGAGATGGCAACCCGCGCGCAGCC-3 Hitchin, BioLabs, h rgn DA(C191 abig isine yPRuigthe using from PCR by amplified Bioscience) Phusion was Cambridge (SC319901, NM_052844.3) cDNA (RefSeq: Origene the gene WDR34 human The stable and lentiviruses lines of cell generation mGFP-WDR34, of Cloning etvrlprilso LXPr-GPWR4wr rdcdin produced were pLVX-Puro-mGFP-WDR34 of particles Lentiviral and I TM ora fCl cec 21)17 7448 doi:10.1242/jcs.159038 4774–4787 127, (2014) Science Cell of Journal Eco nla hte CE12i rsn samnmro dimer or monomer complex. a the as within present is remains TCTEX1D2 It whether complexes. 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O’Connell, T., K. Vaughan, Y., C. Tai, L., D. Dujardin, E., N. L. Faulkner, S. Reck-Peterson, and K. Tan, J., M. Egan, S. R. Patel-King, A., Harrison, W., H. Tedford, E., S. Benashski, M., L. DiBella, ihtann.W r seilygaeu oMr zzlu n alCro (both Curnow help Paul and their Szczelkun for Mark Jepson to grateful Katy especially and are Leard of Wolfson We training. Alan University Bristol with Jepson, the of Mark and University and MRC the Facility, the maintaining Bioimaging Foundation, and Wolfson establishing the for thank Bristol to like would We Acknowledgements with prepared experiments. statistical independent were resulting three images and from data All arise All analyses Illustrator. 4. Adobe version post-hoc and Photoshop Dunn’s Prism using Adobe with GraphPad compared variance using repeated were of Samples test analysis were each. one-way experiments times Kruskal–Wallis all three the and least at shown, independently are images Representative and data analysis image statistical of quantification and Processing aoeu . olt . Genevie M., Goulet, N., J. Dagoneau, D. Asai, and E. L. Ostrowski, S., P. H. Criswell, Okayama, and A. C. Chen, A. T. Zhou, and M. Shen, Y., Yang, Y., Cai, Katayama, A., M. Weiss, K., A. Townley, L., Maccarthy-Morrogh, D., Asante, References at http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.159038/-/DC1 online available material Supplementary material Supplementary for PMC in Deposited Bristol. of number PhD University release. a [grant the immediate of grant and form research D.J.S.; the a to in and Council, MR/K018019/1] Research G1000383-1/1] Medical number UK [grant the studentship by funded was work This Funding manuscript. the authors of All writing manuscript. the the to wrote contributed and and and data reviewed of analyzed conceived project, D.J.S. data; the analyzed directed and experiments performed N.S. and D.A. contributions Author interests. competing no declare authors The interests Competing formation. gradient to density and sucrose experiments on filtration tips gel with for help Blocker their Ariel for UK) Bristol, Bristol, of University yVlct otae(eso .,Pri-le,Se re,UK). Green, Seer controlled Perkin-Elmer, 4.3, NY) (version Rochester, software (Semrock, Volocity sets 63 by filter cells a IX- emission Olympus the with an and microscope and using excitation widefield temperature, imaged were IX-81 room Cells or at and PBS. 71 in min at twice primary 3 rinsed for diluted then the were water] Technologies), distilled of using in (Life performed 1:5000 each was [4,6-diamidino-2-phenylindole staining after at Nuclear DAPI each performed min incubations. 5 antibody of were in secondary PBS PBS with washed temperature in washes Three BSA then 3% temperature. room using room and blocked at min were min cells 5 30 PBS, for for in washes PBS, X-100 two in After paraformaldehyde Triton PBS. 4% 0.1% with fixed with were permeabilized cells 1E,F, Fig. in images ioi n yolsi yenfunction. dynein cytoplasmic B. and R. mitosis Vallee, and Y. Wang, transport. organelle dynein-driven for family. protein LC8 Chem. the Biol. with J. interaction and M. expression, differential S. Dimerization, King, and mtsn . ue,C,Bua,G,Foi . ec,L tal. et rib- L. short Tecco, and E., dystrophy Flori, thoracic III. G., type asphyxiating syndrome, Baujat, cause polydactyly C., mutations Huber, DYNC2H1 S., Smithson, cells. epithelial ciliated mammalian Sci. in Cell DHC1b J. of expression chain: heavy with dynein cells transforming stably for system DNA. transfection plasmid efficient highly a transfer: midbody. and centrosome the the to localized of is Res. control that NudCL through of ciliogenesis overexpression in J. giantin D. Stephens, protein and dynein-2. matrix J. of Golgi C. localization the Westlake, H., for Suzuki, role J., K. 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