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University 55455, Biophysics, and Biology Molecular col ocse,M 10,USA. 01605, MA Worcester, School, eia col ocse,M 10,USA. 01605, MA Worcester, School, Medical uy Awata zone Junya transition large the and proteins at membrane proteins of soluble trafficking ciliary controls NPHP4 ARTICLE RESEARCH ß 4714 2014 August 8 Accepted 2014; April 16 Received ([email protected]) correspondence for *Author USA. 01605, MA Worcester, School, Medical Massachusetts from evidence abundant 1 is there and cilia), in defects by caused 2002). al., in et together Schuermann 2002; 2002; occur al., al., et et Mollet features 2005; Otto al., et disease (Hoefele syndrome both Senior–Løken pigmentosa; retinitis in one Mutations is 2014); al., 18 and et these (Wolf Renkema date, 2014; reported of al., To been et have 2011). Failler renal 2011; NPHP Hildebrandt, Hildebrandt, chronic for of and genes cause human (Wolf causative genetic kidney children frequent cystic in most of the failure form is recessive that a disease is (NPHP) Nephronophthisis INTRODUCTION zone Transition Flagella, Nephrocystin-4, Cilia, Chlamydomonas, CEP290, WORDS: KEY different define other, An each zone. transition of the of zone independently into domains incorporated transition zone are another transition which CEP290, proteins, the two to these distal Therefore, and protein. close zone, membrane transition flagellar the the to of part distal NPHP4, the of into function incorporated in and the it about studied ciliated nephronophthisis have more cause we in learn To NPHP4 humans. widespread in in blindness is defects (NPHP4) and organisms, nephrocystin-4 protein The ABSTRACT ei .Fogarty E. Kevin eateto eladDvlpetlBooy nvriyo Massachusetts of University Biology, Developmental and Cell of Department 04 ulse yTeCmayo ilgssLd|Junlo elSine(04 2,41–77doi:10.1242/jcs.155275 4714–4727 127, (2014) Science Cell of Journal | Ltd Biologists of Company The by Published 2014. PPadSno–øe ydoeaecloahe (diseases ciliopathies are syndrome Senior–Løken and NPHP 3 imdclIaigGop nvriyo ascuet Medical Massachusetts of University Group, Imaging Biomedical NPHP4 NPHP4 1 ak Takada Saeko , hc noe ehoytn4(NPHP4). nephrocystin-4 encodes which , 3 hayooa reinhardtii Chlamydomonas n ereB Witman B. George and edt ueieNH ye4a elas well as 4 type NPHP juvenile to lead 5 rga nMlclrMdcn,Uiest of University Medicine, Molecular in Program 4 eateto ellrBooy nvriyof University Biology, Cellular of Department nphp4 1,2 2 eateto Biochemistry, of Department lv Standley Clive , nl uatfrsflagella forms mutant -null NPHP4 nphp4 PP sstably is NPHP4 . 1, * lglawere flagella uain in mutations . 3 0kDa 50 alF Lechtreck F. Karl , soitdpoen G- n RM1,wihnral are 2011). normally al., et which (Williams cilium TRAM-1a, the from and excluded RGI-2 proteins associated the connect that membrane In (Y-links) overlying 2011). Dupuis-Williams, the links and to (Fisch and microtubules Y-shaped doublet of (Rosenbaum zone zone to transition presence transition compartment cilium the the the of ciliary feature of is conserved base highly the the A 2002). at to Witman, exist access to proposed control gate 2011). or al., ‘pore’ et the Williams 2008; al., et (Winkelbauer cilia Jauregui sensory 2005; the al., of zone et transition the to localize to h aemtn r tne,mshpnadhv axonemal have and misshapen stunted, cilia- are of in Cilia mutant defects 2005). mild same al., has et the NPHP-4 (Winkelbauer for functions al., null sensory et dependent (Wolf mutant cilia a sensory and by 2005), mediated male is in which defects behavior, causes mating NPHP-4 In of body knockdown 2011). (RNAi)-mediated abnormal al., (Burckle including cysts et pronephric phenotypes, and ciliary curvature zebrafish, In classic 2011). exhibit al., et sperm (Won as well development as maintenance A and phenotype. formation photoreceptor ciliary normal a causes NPHP4 of of study loss that systems model o hsfnto.NH4as ih eivle nti activity; this in involved be important the might is of also CEP290 cilia NPHP4 that function. indicate they this and for cilium, evidence and particle the IFT experimental into and Ishikawa protein entry control provide to functions 2008; zone results transition the Rosenbaum, that These and 2011). (Pedersen Marshall, cilia build to flagella organisms intraflagellar all in nearly and defects by used has is also which mutant (IFT), transport The composition 2010). protein al., flagellar et abnormal (Craige in resulting transition another protein, CEP290, of zone absence the in disrupted partially are transition the In included 2005). In which al., 2011). et bodies, (Keller al., basal zone et of Won study 2012; proteomic al., reinhardtii et Patil Chlamydomonas 2005; cells photoreceptor al., – mouse et cilium of connecting – the (Roepman the zone transition in reported elongated and greatly renal 2010) have a mouse al., cultured et Others of (Shiba zone cells 2011). transition the epithelial the in al., in specifically NPHP4 et cilium, of presence primary (Sang the zone of base cell–cell transition of the sites at Several at and present 2012). is zone. contact Yokoyama, cells transition al., IMCD3 the and in to et NPHP4 localizes (Shiba LAP-tagged Reiter NPHP4 that proper 2012; reported have Shah, cilium studies and the Czarnecki and 2012; body between region basal specialized a the zone, transition ciliary the at NPHP4 2008). al., et (Jauregui defects ultrastructural vdnei cuuaigta h rniinzn ucin as functions zone transition the that accumulating is Evidence hs hntpsms ieyrsl rmdfciefnto of function defective from result likely most phenotypes These Nphp4 1,4 .eeasnphp-4 elegans C. alD Bellve D. Karl , mtn iehssonta pp sncsayfor necessary is Nphp4 that shown has mice -mutant anradtselegans Caenorhabditis PP a dniidi a in identified was NPHP4 , .elegans C. uatacmlt h membrane- the accumulate mutant ´ 3 rgr .Pazour J. Gregory , .reinhardtii C. PP a enfound been has NPHP4 , ´ ta. 01 Slanchev 2011; al., et N interference RNA , nphp4 hs links these , morphants 5 ,

Journal of Cell Science oprmns ti ieyta h aiu phenotypic various defects the barrier. to zone due transition mislocalization that the cytoplasmic protein in from follow likely and all organisms flagellar of is membrane- the and consequences crucial It soluble between a both transition compartments. of the is proteins at movement NPHP4 functions the cytosolic associated that control that gate to large indicate wild- selective zone the from results of many excluded The component are flagella. contained normally type that amount; flagella proteins in decreased housekeeping the greatly are were that flagella conversely, proteins membrane-associated wild-type the in of present subset a that found lrsrcue eioae h lglao the axonemal of We and flagella turnover. zone the isolated transition rapid We normal undergo ultrastructure. has generally not and does flagella zone an NPHP4 CEP290, transition identified to contrast the the In to CEP290. close at zone, to transition distal distal and the of membrane NPHP4 periphery that the found at We located organisms. is other and humans to applicable studying from conclusions EERHARTICLE RESEARCH soito ihtefaelm n atof part a and flagellum, the with association entire a slows identify but To motility cell on assembly effects flagellar minor has loss NPHP4 RESULTS hssuy t lglacnb sltdt eemn the determine to isolated highly is be NPHP4 NPHP4. can of ( loss flagella conserved of for its consequences relevant biochemical Particularly study, components. ciliary this and cilia overall affects studying used loss NPHP4. have about its we more how study, learn nor this In zone composition. transition ciliary the in NPHP4 of PP- lotcmltl etrdlnaiy(i.1C), (Fig. of absence linearity the to due restored is phenotype the completely that confirming almost wild-type the of the Transformation NPHP4-R was terms). with material linearity of supplementary definition mutant see but for 1C; (Fig. S1A VCL), the mutant Fig. the S1B, for and lower Fig. much material cells this speeds (supplementary wild-type Swimming assess 2000). To (Mortimer, sperm of 1B). 1C) computer-aided more (Fig. (Fig. using (CASA) slightly cells analyzed analysis are wild-type was paths motility of swimming objectively, those their than but erratic shown), not (data nphp4 for rescued DRC3, fragment transformants, DNA of of a the with some absence transformed and then containing strain, the were wild-type progeny by a a mutant to the compromised twice be backcrossed not to was studies the B1179 was shown in al., analyzed follow et later being phenotype (Lin that the was as complex that ensure and regulatory study To nexin–dynein 2011). 2005) proteomic the flagellar al., of our component et in (Pazour identified FAP134 first was DRC3 mutation, the that revealed pairs PCR primer by termed with analysis PCR Further real-time amplified. by to screened Pazour 1995; was specific al., 1998) et al., (Pazour mutants et insertional of collection our ept hspors,w tl ontko h rcs location precise the know not do still we progress, this Despite The NPHP4 strain. nphp4 nphp4-1 .reinhardtii C. NPHP4 mtn el r oieadcnudrophototaxis undergo can and motile are cells -mutant .rihrtinphp4 reinhardtii C. NPHP4 ee eea rdce ee hthv oknown no have that genes predicted several gene, deleted , DRC3 NPHP4 noesri B19,n rdc was product no (B1179), strain one In . nl uat h uathsfull-length has mutant the mutant; -null ee(i.1) n fteresulting the of One 1A). (Fig. gene , ohmnBAT value E BLASTP human to .reinhardtii C. .reinhardtii C. 0kparound kbp 40 DRC3 uain nhmn n other and humans in mutations NPHP4 a sdi hssuya the as study this in used was , nphp4 uat eoi N from DNA genomic mutant, eet eeaestrain generate to gene a ayavnae for advantages many has PP r ieyt be to likely are NPHP4 uatwr similar were mutant NPHP4 nphp4 DRC3 .reinhardtii C. nldn the including , 5 uatand mutant 1e Fg 1A). (Fig. NPHP4 2 nphp4 85 ,so ), to . pcfct PP.Smlry h niH nioylocalized antibody In anti-HA the 1F). but (Fig. Similarly, cells NPHP4. wild-type cells to of flagella specific antibody rescued the of anti-NPHP4 the bases not the the labeled in strongly microscopy, specificity cells, immunofluorescence with protein high NPHP4–HAC tagged mutant the and recognized with the C-terminus antibody NPHP4–HAN or anti-HA rescued N- generate the the respectively; also to either We at NPHP4 tag of mutant. 3xHA a the NPHP4 expressing that constructs in confirmed results missing These wild- is 1E). of (Fig. antibody blots strain western The in NPHP4-R not size NPHP4. expected but of the type of C-terminus band the a recognized against antibody in located an is NPHP4 zone where transition determine the To in located is NPHP4 PP–A el.A xetdfo h immunofluorescence the from and expected NPHP4–HAN As of cells. wild-type, microscopy NPHP4–HAC from zone, immunoelectron transition apparatuses pre-embedding the nucleoflagellar out within NPHP4 carried of we location the transition determine distal To the of periphery the zone at located is NPHP4 is NPHP4–HAC zone. of peak-to-peak transition C-terminus mean , the CEP290 the the and that CEP290 the in between indicated distance located to images TESM is distal of fluorescence/epi- NPHP4 be Analysis that to reflection indicating observed the label, 3C), was internal (Fig. label (TESM) microscopy NPHP4–HAC microscopy total light immunofluorescence structured and fluorescence wide-field al., 3A,B) both et (Fig. (Craige NPHP4– zone By transition peptide the the HA in 2010). the located of against is which antibodies CEP290, cells with and labeled whole were strain and HAC apparatuses that nucleoflagellar flagella. indicate the NPHP4–HAC in results in not detected the but The body, be were basal 2D). could of (Fig. none flagella flagella all and reinhardtii isolated body isolated this blotting; cell and confirm the the To western bodies within 2C). remained (Fig. cell by bodies cells, cell compared were the whole and with was result, cells bodies NPHP4–HAC stay anti-HA; cell to with NPHP4–HAC resulting found stained the were 1989). flagella and detached dibucaine flagella proper Salisbury, axoneme with the and deflagellated zone deflagellated, and transition are the of (Sanders cells junction the cells When carefully at detach we kidney bodies. in Therefore, cell mouse NPHP4 2012). of al., versus and distribution et MDCK the Patil determined 2005; of al., cilia et (Mollet primary to 2B). localize (Fig. the and NPHP4 that HA-tagged NPHP4–HAN indicating to both cells, specific wild-type in is in localization flagella not but of cells bases NPHP4–HAC the to specifically hw) olwn lgla muain the amputation, not data 2A; flagellar Fig. 1D; Following (Fig. normal shown). ultrastructurally be to steady-state appear normally, nearly of swim to flagella ability the with Consistent PP- tan(i.1) hrfr,NH4i eddt build the to kinetics. in needed normal is with restored NPHP4 flagella Therefore, the completely cells; 1D). (Fig. wild-type was strain did NPHP4-R regeneration than flagellar slowly more normal flagella new regenerated 3 m( nm 137 ofrhrnro ontelcto fNH4 both NPHP4, of location the down narrow further To to microscopy immunofluorescence by found been has NPHP4 nphp4 ora fCl cec 21)17 7442 doi:10.1242/jcs.155275 4714–4727 127, (2014) Science Cell of Journal n PP slctdi ihrtetasto oeo the or zone transition the either in located is NPHP4 nphp4 el Fg A,idctn htti oaiainis localization this that indicating 2A), (Fig. cells 5 nphp4 130). uatclsaeo ernra eghand length near-normal of are cells mutant hl el;tebn a etrdi the in restored was band the cells; whole .reinhardtii C. .reinhardtii C. nphp4 emade we , flagella mutant 4715 C.

Journal of Cell Science fwoeclspoe iha nioyaantteCtriu fNH4 h nioyrcgie ad(roha)o h rdce iefrNPHP4 for size predicted the of (arrowhead) band a recognized wild-type, antibody of The blot Western NPHP4. (F) of strain. NPHP4-R C-terminus the the and against cells wild-type antibody in an kDa) with (210 probed cells wild-type, whole of deflagellation of after time of function a upeetr aeilFg 1.Frec tan h ouainwscluae rmtesmo ausfo oa ftnfed nec ffv indepen five of each peptide. in HA fields the ten against of antibody total a from values of sum ** the method: wild-type, from Tukey–Kramer the of calculated the to populations was by attachment of population analyzed determined by paths the not was immobilized swimming strain, were significance were in each tracks Statistical dots linearity For Blue red experiments. of S1). analyzed. with distribution Fig. paths marked the material swimming Cells summarizing supplementary the completed. (right) are was histogram lines recording and green before of linearity tracks; field paths their microscope swimming monitor the The to outside coverslip. began swam CASA cells where the cells because show dots White cells. ogn D nPyooevrin91(tp/wwpyooent.Arw niaetepstoso C rdcsue odlmttedltdregion; k for deleted used the clones delimit (BAC) chromosome to artificial used bacterial products from fragments PCR Genome of amplified. positions were the products PCR full-length indicate the of Arrows whether in (http://www.phytozome.net). indicate marks 9.1 minus version and Phytozome plus in IDs gene to EERHARTICLE RESEARCH 4716 the of Characterization 1. Fig. NPHP4 and DRC3 .rihrtinphp4 reinhardtii C. b r niae ywiercage.W,wl ye B ersnaieiae hwn wmigptso idtp and wild-type of paths swimming showing images Representative (B) type. wild WT, rectangles. white by indicated are nphp4 1APs a sda odn control. loading a as used was F1-ATPase el r oeertcta hs fwl-yecls cl a:50 bar: Scale cells. wild-type of those than erratic more are cells nphp4 mutant. n PP- el.Faelrlnt tec iepiti h mean the is time-point each at length Flagellar cells. NPHP4-R and A a of Map (A) .reinhardtii C. nphp4 P eoena the near genome PP–A n PP–A hl el rbdwt monoclonal a with probed cells whole NPHP4–HAC and NPHP4–HAN , , .1 *** 0.01; ora fCl cec 21)17 7442 doi:10.1242/jcs.155275 4714–4727 127, (2014) Science Cell of Journal P , .0.()Fael eghpirt elglainadas and deflagellation to prior length Flagella (D) 0.001. NPHP4 m .()Brgah(et hwn h mean the showing (left) graph Bar (C) m. ou.Nmesaoeec ou correspond locus each above Numbers locus. nphp4 6 ..o 0fael.()Wsenblot Western (E) flagella. 30 of s.d. n PP- el (see cells NPHP4-R and 6 nphp4 ..of s.d. nock- dent

Journal of Cell Science EERHARTICLE RESEARCH oe fattlo 0gl atce rmlniuia etosof sections longitudinal from particles gold 50 of total a of zone, labeling immunogold NPHP4. the wild- HA-tagged Witman, the that to specific with and indicating was associated zones, (Kamiya particles transition gold detergent type no observed nonionic We with 1984). resistant is treatment which membrane, association longitudinal zone to close transition zone in the periphery, in transition of zone remnants the with transition of particles the NPHP4–HAC cross-sections at located in gold and were particles all NPHP4–HAN Most and both sections 4A,B). (Fig. in with zone specimens associated were transition particles gold the 2B), (Fig. results microscopy h oain,rltv otepoia n ftetransition the of end proximal the to relative locations, The E20 h vrg itnebtenNH4HCand the with NPHP4–HAC agreement good in between nm, ( 111 distance separation was particles average gold CEP290 the CEP290; 155 atce a hr ek ntedsa rniinzn.The the zone. of transition end proximal 173 distal gold the was the from our zone NPHP4–HAC particles in transition gold with and of peaks the Consistent distance Craige sharp average NPHP4–HAN 4C). 3), by had (Fig. (Fig. both particles 2010) CEP290 of observations al., for distribution microscopy et determined (Craige immunofluorescence that al. to et compared measured were specimens and NPHP4–HAC and NPHP4–HAN both 6 1n o PP–A,cmae o61 to compared NPHP4–HAN, for nm 31 ora fCl cec 21)17 7442 doi:10.1242/jcs.155275 4714–4727 127, (2014) Science Cell of Journal , 3 m esrdb TESM. by measured nm) 137 6 7n ( nm 37 oddprcl body. cell per loaded that indicate control. loading a was as IC2 used protein axonemal anti-HA the the antibody; with a probed on blot analyzed western were flagella bodies isolated cell and cells, whole NPHP4–HAC 2 (D) bars: Scale peptide. HA labeled against and antibodies fixed with cell body NPHP4–HAC cell an and of flagella the Detached on (C) are right. images merged anti-HA; tubulin and anti-acetylated with labeled were cells Wild-type, NPHP4–HAC (B) and right. NPHP4–HAN the on are images merged NPHP4; flagellar and for microtubules) marker (a against tubulin antibodies acetylated with labeled were cells base the to flagella. localized of is NPHP4 2. Fig. 6 .. o PP–A and NPHP4–HAC for s.d.) A idtp W)and (WT) Wild-type (A) , ,5o 0fael ar were pairs flagella 20 or 5 1, a tblnadthe and -tubulin 6 m 6 1, m. 6 8n for nm 38 and 5 nphp4 4717 6 20

Journal of Cell Science EERHARTICLE RESEARCH odtriewehrls fNH4cue structural caused NPHP4 transition 4718 the of compared we loss zone, transition the whether in abnormalities determine the to in zone To transition contrast the of in zone. Ultrastructure was transition Therefore, NPHP4 the at HA-tagged NPHP4. static any, is NPHP4 wild-type if CEP290, at as little, with min that 60 replaced at indicating bright were as was min, zones label 20 over the zones HA-tagged Moreover, transition transition period. any, wild-type time two if this the little, into only that incorporated was indicating min, NPHP4 anti-HA antibody, 60 with the labeled after by labeled and cytoplasm. Even reaction min 60 mating 5). common and (Fig. the 40 a of 20, initiation in at and after prepared NPHP4 NPHP4 were untagged apparatuses HA-tagged Nucleoflagellar contained contained zones transition two two zygotes NPHP4– quadriflagellated which with form in is to gametes is gametes NPHP4 wild-type NPHP4–HAC if zone or mixed determine HAN transition To we the 2010). dynamic, al., at similarly et CEP290 (Craige dynamic that highly previously the reported rapidly over We into turn not assemble does and NPHP4 to other. each transported of independently are zone transition proteins two these nphp4 the in normal be to cep290 appeared we NPHP4 of Localization versa, of respectively. vice microscopy and immunofluorescence nphp4 CEP290 out of carried independently zone transition if explore To zone other transition each the of to independently localize CEP290 and NPHP4 mtn el ihatbde gis PP n CEP290, and NPHP4 against antibodies with cells -mutant el splmnaymtra i.SAB.Therefore, S2A,B). Fig. material (supplementary cells el,adlclzto fCP9 a omli the in normal was CEP290 of localization and cells, .reinhardtii C. PP a easmlda the at assembled be can NPHP4 nphp4 cep290 mutant and - aeilrsmln h eta yidr fthe of 6C). cylinders (Fig. zone in central transition dense the of Finally, localization resembling ectopic apparently same. material the zone, transition the the to not of sections are longitudinal structures present are Y-links shaped the the because Thus, the Moreover, NPHP4. in S2C). of related loss specifically Fig. not the is to material structure this (supplemental in variation morphological flagella of their To microscopy of 2007). immunofluorescence al., performed lack et ift46 with we (Hou correlated is NPHP4, IFT46 structure of this protein of IFT disruption whether the zones clarify transition lacking in cells altered of similarly are structures wedge-shaped oedult n ebaei idtp el,apae obe to appeared cells, the the in wild-type collapsed sections, in transition or missing membrane the longitudinal and between the doublets In extend zone 6A). which between (Fig. connectors, normal wedge-shaped Y-links appeared are which the and membrane, the and sections, in microtubules disrupted doublet cross zone transition In microscopy. oe fwl-yeand wild-type of zones ifrne eeosre ntemmrn-lsmti fractions. membrane-plus-matrix the in observed the were between differences of observed were gels and differences SDS-polyacrylamide wild-type few In very fractions, fraction. axoneme axonemal fraction an membrane-plus-matrix separate and detergent-soluble to a NP-40 with into treated versus proteins and wild-type isolated were of flagella compositions flagella, protein the NPHP4 compare is of To matrix loss and by membrane altered flagellar drastically the of composition Protein el n on htNH4wsnral oae ttebase the at located normally was NPHP4 that found and cells nphp4 ora fCl cec 21)17 7442 doi:10.1242/jcs.155275 4714–4727 127, (2014) Science Cell of Journal uat ecncnld htte n h wedge- the and they that conclude can we mutant, nphp4 cep290 ape Fg AB.Hwvr numerous However, 7A,B). (Fig. samples uat(riee l,21) eepresent were 2010), al., et (Craige mutant nphp4 nphp4 nphp4 lgla eosre,js distal just observed, we flagella, 2 bars: two Scale show examples. C additional in insets lower panels; the the in shown the zones of transition inset enlargements upper are the C and in B and A The in C). insets in only separate (shown from right channels merged the are in C image of the panel in and images B the and All A (C). TESM or (A,B) microscopy immunofluorescence widefield by conventional acquired were Images and CEP290. peptide HA tubulin, acetylated against antibodies with labeled were (B,C) cells whole NPHP4–HAC or (A) cells NPHP4–HAC of apparatuses nucleoflagellar the to zone. localized transition is NPHP4 3. Fig. uat(i.6) oee,the However, 6B). (Fig. mutant m m. mtn el yelectron by cells -mutant Isolated .reinhardtii C. , 0 of 30% nphp4

Journal of Cell Science EERHARTICLE RESEARCH ifrne eedet oso PP.Ipraty most Importantly, NPHP4. of and wild-type loss the to between due the differences in that were confirming restored S3), was differences Fig. composition (arrows, material protein (supplementary increased flagellar NPHP4-R were Normal others 7A). the whereas Fig. in 7A), decreased Fig. were proteins (arrowheads, some of levels The hntepoenwsntfudi h te lc wl yeor type (wild slice other five the than in greater found be not to nphp4 was the type, had protein wild recovered used the to peptides we compared when of ten number flagella, than the be more mutant to or by had the mass recovered decreased peptides or in of by increased number slices altered the (1) analysis was all criteria: amount following protein the to in that a conclude identified subjected To of S1). were Table then material proteins (supplementary were Multiple slices spectrometry. The wild- S3). of fraction membrane-plus-matrix the type, of gels from excised 7B). (Fig. nphp4 kDa kDa; 50 50 above below range observed mass were the differences in few observed were fractions matrix oietf h rtista eedcesdo nrae nthe in increased or decreased were that proteins the identify To nphp4 en oprd n 2 h hnewsspecifically was change the (2) and compared; being ) lgla eso lcscnann h icratbnswere bands discordant the containing slices of sets flagella, n PP- lgla(upeetr aeilFig. material (supplementary flagella NPHP4-R and nphp4 membrane-plus- nphp4 mutant 05.Alicesdpoen r rdce ob agrthan larger proteins be housekeeping large in to that increased conclude predicted specifically We are 1). are (Table proteins kDa 50 increased All synthetase, not 2005). the were phenylalanyl-tRNA protein in and RNA-binding found Valyl- and bodies. ribosylglycohydrolase ADP cell font in the abundant red are increased presumably in that that or proteins proteins housekeeping all Except were A, blue amount respectively). complex-subunit V1 proteins, (where ATPase decreased for S1 or the increased Table criteria, indicates amount these material in decreased on or supplementary the increased Based be to cells. in found were NPHP4-R that the proteins in rescued rtisas rdce ob ebaeascae u not in all at but detected not less associated were by flagella, identified wild-type membrane in were peptides other proteins be five (these than examined criteria to above the we and predicted meeting when domain, also N- transmembrane Additionally, have proteins FAP12 a sites. and FAP295 have myristoylation FAP33, kinase, to calcium adenylate the predicted flagellar – associated is membrane be ATPase to predicted are FAP208) ycnrs,alpoen htdcesdi mut(te than (other amount in decreased that proteins all contrast, By ora fCl cec 21)17 7442 doi:10.1242/jcs.155275 4714–4727 127, (2014) Science Cell of Journal nphp4 .reinhardtii C. mtn lglaaelse nTbe1and 1 Table in listed are flagella -mutant nphp4 lgla rtoe(aore al., et (Pazour proteome flagellar vsbei h ogtdnlsection) longitudinal the in (visible the the of on scale the to example matched typical a electron shows The micrograph each. for scored particles were gold 50 zones; transition NPHP4–HAC and NPHP4–HAN of sections longitudinal with particles associated gold of Distribution (C) membrane. the zone with transition association close transition in the zone, of periphery the at were found particles gold All (arrowheads). particles gold with labeled zones transition (c,d) and NPHP–HAC (a,b) NPHP4–HAN of sections Cross- (B) (arrowheads). gold particles with labeled zones transition (c,d) NPHP4–HAC and (a,b) of NPHP4–HAN sections longitudinal Typical particles. (A) gold 10-nm to conjugated anti-rat-IgG by with followed incubation peptide, HA directed the IgG against rat with incubated were cells NPHP4–HAC or NPHP4–HAN of apparatuses nucleoflagellar the zone. in transition located distal is NPHP4 4. Fig. Cag ta. 00.Saebars: nm. Scale 50 2010). al., al. et et (Craige Craige from of replotted distribution CEP290 the show bars The green N-terminus. the than more distally slightly located be might of NPHP4 C-terminus The the zone. and transition body basal the between mtn flagella. -mutant y ai niae h boundary the indicates -axis Isolated y ai;‘0’ -axis; 4719

Journal of Cell Science nyi-eetpoen(FAP33) protein Ankyrin-repeat EERHARTICLE RESEARCH 4720 1 bars: Scale images. in merged in the increased Decreased in or shown st decreased zones and Proteins transition fixed the prepared, 1. were Table of zygotes enlargements quadriflagellated are resulting panels the right of The apparatuses DAPI. nucleoflagellar and mixing, anti-HA after tubulin, times acetylated static. indicated is the zone transition At the reaction. at NPHP4 5. Fig. rgyeielps (FAP12) lipase Triglyceride CaATPase rnmmrn domains; transmembrane nyi-eetpoen(A28 9kaMtinn ytae8 kDa 87 synthase Methionine identifie peptides of number the in differences on based selected were proteins listed The versus analysis. wild-type the spectrometry comparing versus mass gels SDS-polyacrylamide wild-type to in subjected amount and in excised changed were drastically were that bands kDa Protein 99 (FAP295) kinase protein cA(G)MP (FAP208) protein Ankyrin-repeat (FAK1) kinase adenylate Flagellar b nphp4 nphp4 lglaIcesdin Increased flagella c mtn ape,uigciei ecie ntetext. the in described criteria using samples, -mutant a rtisntfudi h lgla rtoesuy(aore l,2005). al., et (Pazour study proteome flagellar the in found not proteins a a a idtp W)gmtswr ie ihNH4HN()o PP–A B aee oiiit h mating the initiate to gametes (B) NPHP4–HAC or (A) NPHP4–HAN with mixed were gametes (WT) Wild-type nphp4 1kaHa hc rti 08 kDa 81 kDa 135 protein binding RNA 90 protein shock Heat ribosylglycohydrolase ADP 3 factor Elongation synthetase Valyl-tRNA kDa 52 kDa 81 kDa 123 kDa 173 kDa 213 flagella a rtiswt rdce -yityainsite; N-myristoylation predicted with Proteins yy-RAsnhts 7kDa 67 kDa 76 kDa 68 kDa 68 synthetase Phenylalanyl-tRNA synthetase Lysyl-tRNA ligase A Formate-tetrahydrofolate subunit complex, V1 ATPase, synthetase Glycyl-tRNA ora fCl cec 21)17 7442 doi:10.1242/jcs.155275 4714–4727 127, (2014) Science Cell of Journal nphp4 flagella c c nphp4 c c lgla ebaepu-arxfractions membrane-plus-matrix flagellar b rti ihpredicted with protein 9kDa 59 kDa 77 kDa 94 kDa 141 m ie ihanti- with ained m. nthe in d

Journal of Cell Science EERHARTICLE RESEARCH h eeso oto hs rtisddntse ob lee in altered be to seem not did proteins and these spectrometry, of mass most by of identified levels the flagellar were proteins IFT the Several with the in a NPHP4. IFT associated only of absence that the normally conclude in decreased We proteins is flagella. membrane it of mutant that the confirmed subset in blot reduced western The was analysis. spectrometry in decreased mass be not to appeared were which 2007) FMG-1B, al., the et in Huang The polycystin-2; decreased PKD2 dataset. human and well-characterized of our 1996) homolog al., in (a two et (Nakamura identified protein against mastigoneme not flagellar proteins antibodies membrane the with and flagellar wild-type cells from flagella in mutant isolated amount of blots in western not reduced are are NPHP4. or of proteins absence into the transported in flagella not the either in the retained with are were associated N- normally membrane amount proteins flagellar Thus, in 2005). contain material decreased al., the et that (Pazour in supplementary which proteins identified in all previously Nearly apyrase, font S1). and (yellow – Table and domains sites transmembrane have myristoylation protein ion which of transporter predicted all ankyrin-repeat ABC – an a FAP29 ATPase, and transport large FMG1-B, calcium the another glycoprotein included channel, These membrane flagella. mutant flagellar the in decreased were the odtriewehralfaelmascae membrane flagellum-associated all whether determine To nphp4 nphp4 uatadwr etrdi h ece tan,all strain), rescued the in restored were and mutant nphp4 mutant lgla(i.7) eas rbdfor probed also We 7C). (Fig. flagella .reinhardtii C. nphp4 nphp4 lgla eprobed we flagella, lgla proteome flagellar lglai our in flagella nphp4 - lgtydcesdi h bec fNH4(supplementary was NPHP4 velocity of IFT absence whereas S4C). the Fig. affected, in material not S4B). decreased is Fig. steady- was slightly IFT material in frequency (supplementary whether movement analyzed IFT IFT-particle assess was NPHP4, directly flagella of or state more lack unaltered To by were affected BBSome, S4A). the Fig. the of material in and subunit A a elevated complex both BBS4, slightly of of proteins IFT in and of To B, components motors, IFT IFT S1). of of levels The Table and amounts wild-type the of material flagella examined isolated (supplementary we this, flagella confirm mutant the oan ftetasto oe(i.7) Immunoelectron 7D). (Fig. zone structural transition distinct the define CEP290 two of the and between domains NPHP4 overlap little Thus, is of proteins. there minimum that revealed a microscopy were to by TESM distal by zone, observed transition separated peaks the CEP290 of and NPHP4 part The immunofluorescence distal CEP290. the in both located that is determined Using NPHP4 we that microscopy, NPHP4. immunoelectron found and of microscopy we distribution localization the 2011), on information and al., precise more much et provide studies Williams 2011; 2010; reinhardtii al., al., et Shiba et 2008; al., Sang et Jauregui 2005; in al., studies et (Winkelbauer previous with agreement In the of regions zone distinct transition define CEP290 and NPHP4 DISCUSSION ora fCl cec 21)17 7442 doi:10.1242/jcs.155275 4714–4727 127, (2014) Science Cell of Journal PP slctdi h rniinzn.Hwvr our However, zone. transition the in located is NPHP4 h omltasto oeclnes(ros.Saebars: nm. Scale 50 (arrows). resemble cylinders structures zone transition these normal zone; the transition the just to (arrowheads) distal structures cylindrical ectopic contain rniinzns C bu n-hr of one-third About the (C) in zones. obscured transition but or zones, deformed transition missing, NPHP4-R are and wild-type in are present (arrowheads) structures wedge-shaped opaque of zones transition wild-type, through sections Longitudinal the (B) in normal appear (arrowheads) and (WT) wild-type the in structure mutant. zone Transition 6. Fig. A rs-etostruhtasto oe of zones transition through Cross-sections (A) nphp4 nphp4 n PP- el.Teelectron- The cells. NPHP4-R and mtn lgla(supplementary flagella -mutant , nphp4 nphp4 4 m n immunoelectron and nm, 140 .elegans C. el.TeY-links The cells. el ywsenblotting. western by cells nphp4 nphp4 n mammals and nphp4 nphp4 mutant. flagella 4721 C.

Journal of Cell Science EERHARTICLE RESEARCH ihtoeo age l Sn ta. 01,wocnlddthat concluded who 2011), 4722 al., et (Sang agree al. findings et These Sang zone. of those transition with the indicate in results complexes assembly different and absence localization the the in that Both zone other. transition the CEP290 the and of into NPHP4 that normally found assembled we each zone, transition the of regions proper. flagellum no the is in there Finally, that NPHP4 indicate microtubules. detectable flagella isolated and of membrane studies 2010); biochemical the our between al., extends NPHP4 space of et the part middle into (Craige the that out the membrane rule of cannot between the we that however, space and with the microtubules throughout contrasts doublet distributed the zone well proteins is to transition which NPHP4 membrane is the CEP290, of of of localization NPHP4 periphery apparent exit extreme Thus, This and/or flagellum. membrane. entry the from control zone to transition association positioned close in the zone, transition the with are of NPHP4 periphery of the C-termini at and located N- the that indicated also microscopy ossetwt PP n E20biglctdi different in located being CEP290 and NPHP4 with Consistent .reinhardtii C. PP n E20aeitgae into integrated are CEP290 and NPHP4 nsutigITo te iir rtisit h rniinzone. transition the into highly proteins ciliary role Although other a or dynamic. has IFT CEP290 shuttling that more contrast, possibility in the By much raise protein. results be the structural speculative, to a transition span for the appears time expected at CEP290 same be static the might is NPHP4 in as Therefore, zone, replaced 2010). to 50% al., contrast least sharp et in (Craige at is is this period; which 1-h a CEP290, during turnover no or little proteins distinct lysates. LAP-tagged of whole-cell of components from immunoprecipitation were on based CEP290 modules, and NPHP4 mammalian ig,16) riee l Cag ta. 00 bevdta the that observed 2010) 1986; al., al., et et (Craige al. Perkins et 1972; Craige Satir, of 1967). Ringo, and cross-sections (Gilula microscopy and organisms electron microtubules various in doublet is observed the zone as between transition membrane Y-links the of of feature occurrence defining the and conserved highly A for consequences structure different zone have transition loss CEP290 and NPHP4 eas bevdta PP ttetasto oeundergoes zone transition the at NPHP4 that observed also We ora fCl cec 21)17 7442 doi:10.1242/jcs.155275 4714–4727 127, (2014) Science Cell of Journal ersn rniinfbr oncigtebslbd to body membrane. triangles basal plasma Gray the the zone. connecting transition fibers the transition of represent end membrane proximal and the the microtubules at in doublet located the of is between end (green) space distal CEP290 the zone. at in transition membrane located the the is with (red) NPHP4 association zone. close and transition NPHP4 the of in distribution CEP290 the control. illustrating loading Diagram a arm (D) as outer served The IC2 chain PKD2. intermediate and dynein protein mastigoneme the 1B, h uatfael.()Wsenbo fioae wild-type isolated of blot Western and (C) in flagella. amount mutant in increased the that proteins some indicate arrows in the decreased in that amount proteins some indicate fractions. Arrowheads versus axonemal (WT) and wild-type (M+M) compare membrane-plus-matrix to used were gels composition. of Flagella 7. Fig. nphp4 lglapoe ihatbde gis FMG- against antibodies with probed flagella nphp4 %()o 2 B SDS-polyacrylamide (B) 12% or (A) 6% nphp4 ebaepu-arxfraction; membrane-plus-matrix el aeanra protein abnormal have cells nphp4

Journal of Cell Science ifso are per ob oeiprebet the to impermeable more be proteins. soluble the to large of old, appears entry that diffusion-based h flagella barrier 3 steady-state the least diffusion in Because were at barrier.’’ observed diffusion were the we by that is regulated differences cilia be in proteins to of unlikely distribution steady-state ‘‘the that suggested -ik eemsigo irpe in disrupted or missing were Y-links ARTICLE RESEARCH ifso are ttebs fpiaycla oee,they However, cilia. as large primary size-dependent as of proteins a of base for entry evidence the slow observed obtained at also barrier 2013) diffusion the al., in et to NPHP4 (Lin protists upon from soluble dependent is ranging of barrier organisms diffusion zone. this transition of to that cilia barrier and humans the size-dependent into a proteins is indicate there results these together, that Taken proteins. larger for observed were the proteins many of of all amounts the The nearly in increased fraction. Strikingly, axonemal fraction, flagella. the membrane-plus-matrix the not the in were for observed of differences NPHP4 consequences biochemical of the assess loss to which able with were we ease purified, the of soluble in large Because breached of is entry proteins ciliary housekeeping to barrier size-dependent A rtisicesdi iefo tksrdu f31n to nm with 3.1 of working radius the Stokes of as to 2013), a entry slowed (corresponding from of progressively al., size nm rate cilium 4.3 in the the et increased Similarly, that into proteins not. (Breslow found proteins did cells, soluble al. IMCD3 latter permeabilized et the whereas kDa) the Breslow (67 entered compartment, (BSA) former the albumin cells, ciliary serum RPE hTERT labeled bovine into fluorescently and microinjected when were kDa) that (41 observed A 2012) excludes al., protein et that (Kee al. zone et transition of an the proteins is soluble there at that indicating barrier SDS-PAGE, in NPHP4-dependent kDa 50 above migrated the into organelle. keeps wild-type the normally that of from the mechanism out a them in of fraction breakdown presence the membrane-plus-matrix from their resulted that the suggesting detected in not flagella, were all soluble proteins were these proteins at of these of Most all proteins. V1-ATPase, housekeeping the of A subunit of Y-link for in required true not is is same NPHP4 The affect Therefore, not formation. did contrast, Y-links. by NPHP4, of the loss that found We mutants. eels Wlim ta. 2011). al., et (Williams members in lost and combined (Jauregui were NPHP4 were Y-links module in MKS/B9 the mutations the when of of However, disruption 2008). cause al., not et did alone NPHP4 raim(un ta. 92 iscie l,20) The moving flagellum. 2008). by the into responds al., precursors and abnormalities cylinder NPHP4 et detects central of additional cell absence Piasecki the the with 1982; because associated occur al., might this et of duplication mutants (Huang uniflagellar in observed organism been has cylinder the central transition of the duplication of of lumen partial the zone a occupies normally be that to cylinder central appeared what zone, transition nphp4 sn hmclyidcbedfuinta prah i tal. et Lin approach, trap diffusion inducible chemically a Using motnl,anra nr fslbectpamcproteins cytoplasmic soluble of entry abnormal Importantly, lhuhn pcfcsrcua bomlte eeosre in observed were abnormalities structural specific no Although rniinzns h lglaotncnand itlt the to distal contained, often flagella the zones, transition .reinhardtii C. nphp4 lglu a bevdol o rtisthat proteins for only observed was flagellum nphp4 , 0kao agr ossetwt hs Kee this, with Consistent larger. or kDa 50 neetnl,asmlrdpiaino the of duplication similar a Interestingly, . lgla n,wt h osbeexception possible the with and, flagella, , .reinhardtii C. 0to 30 .reinhardtii C. .elegans C. , 0ka;n nr was entry no kDa); 70 .reinhardtii C. nphp4 .elegans C. , 0–5 D,and kDa, 600–650 NPHP4-dependent hr osof loss where , nphp4 cells lglacnbe can flagella h Y-links the , flagellum CEP290 ae oehr hs eut ugs htasbe fflagellar in of mobile subset be to a presumed those that specifically of suggest – plane proteins 2013). results the membrane Bloodgood, these in and moved (Guilford together, is al., IFT Taken protein distributed by et this membrane (Pazour of flagellar evenly some the fractions least were axonemal at and and 2005), FMG-1B membrane the from the the between that in peptides observed decreased; further was proteome, We FMG-1B 2011). al., of et odorant level Williams 2005). an 2008; and al., al., PKD2 et et in that this located (Pazour reports normally that axoneme are with receptor well the implying agree to detergent, results anchored to These were is resistant proteome also fraction flagellar protein the the in the in from found peptides identified of 75% protein Similarly, (Pazour mastigoneme 2007). proteome al., et flagellar (Huang flagella the and in 2005) al., fractions were et peptides axonemal PKD2 most in because axoneme found the to anchored be protein, to mastigoneme flagellar in the affected other and not PKD2 some were as of such levels proteins, the membrane However, decreased. proteins oae eaiet PP n E20i h rniinzone. of transition be the are in will proteins CEP290 it and MKS/B9 NPHP4 the so to where relative proteins, precisely located proteins transmembrane the determine MKS/B9 at be to or the interest to to of close from predicted several located and are proteins is membrane NPHP4 membrane zone cilium. transition ciliary the of prevent to 2010; out likely al., diffusing to are al., et proteins together Warburton-Pitt MKS/B9 et 2011; the in function al., and Williams et NPHP4 interact Therefore, Huang 2008; 2012). ciliary 2011; al., al., genetically for et important et Williams complex are (Williams that MKS/B9 development pathways the redundant MKS/B9 functionally the of of in part not proteins studies is the NPHP4 but several 2012). al., to of complex, et amount Chih localizes (Garcia-Gonzalo 2011; the al., cilia in et primary which in decrease proteins a complex, membrane in GFP-tagged MKS/B9 resulted zone, the transition of our disruption of with that consistent showed is members cells that mammalian cilium with the Studies of findings. base the at barrier protein of the to at out barrier free diffuse NPHP4-dependent zone. are an subsequently of transition that membrane absence the the those in of organelle but the plane flagella, the the in proteins to move membrane flagellar normally all targeted or the most are to that that normally possible delivered is protein be it to mastigoneme flagella, appear the axoneme exhibit the and axoneme to PKD2 bind the in to Because levels anchored reduction. lower are no at that present those are whereas – flagella, membrane the of plane the lhuhtelvl flreslbehueepn proteins housekeeping soluble large of in levels increased flagella a the the retains in that Although proteins barrier membrane zone of transition a subset to essential is NPHP4 hc osiue h oiemciey oee,cagsin changes However, machinery. axoneme, Ca motile flagellar intraflagellar the the if of little, constitutes composition is protein there which the that in finding change our with any, consistent the is in defect observed motility we that defect motility nphp4 behavioral flagellar on only effects the mild Interestingly, has NPHP4 of Loss wmigta eosre setrl ossetwt h osof loss erratic the the with so consistent 1984), entirely is Witman, observed and we Kamiya that swimming 1980; al., et (Bessen rvossuishv rvddeiec o membrane a for evidence provided have studies Previous uatwsertcsimn.Telc fastronger a of lack The swimming. erratic was mutant ora fCl cec 21)17 7442 doi:10.1242/jcs.155275 4714–4727 127, (2014) Science Cell of Journal nphp4 2+ . 0 fGPtge K2i ttoayi the in stationary is PKD2 GFP-tagged of 90% r rca o otoln lgla waveform flagellar controlling for crucial are lgla eeso aymembrane-associated many of levels flagella, nphp4 .elegans C. flagella. .eeasnphp-4 elegans C. .reinhardtii C. niaeta PP and NPHP4 that indicate .reinhardtii C. K2i likely is PKD2 ii (Jauregui cilia flagellar nphp4 nphp4 4723

Journal of Cell Science mt+ n 6357-bp a and ( 79D8 ( B1179 MN). Paul, ARG7 St Minnesota, of (University ieiso lgla eeeainfloigfaelramputation flagellar following regeneration flagellar of kinetics zone Although transition functional the and in structural domains different define CEP290 and NPHP4 levels. Ca the as such proteins membrane ARTICLE RESEARCH 4724 second and first three the [containing between a NPHP4–HAN tag) NPHP4], of (3xHA encoding acids peptides amino (Stratagene). construct SK- HA a II consecutive pBluescript generate into sequentially To inserted and NPHP4 full-length the clone To transformation and S2. Green cloning Table Gene a SYBR material supplementary using QuantiTect in PCR listed the primers real-time and Research), by (Qiagen) MJ kit determined PCR (Opticon, was B1179 cycler of thermal site deletion The PCR Real-time 137c, and CC124 with strains progeny wild-type selected transforming the then to and sequentially B1179 crossing nit2 reinhardtii C. medium growth and Strains zone. METHODS transition AND the MATERIALS in domains structural functional as different define well CEP290 as IFT. and in NPHP4 function that additional 2010). conclude an the We has to al., CEP290 essential barrier, et both zone are (Craige transition CEP290 the and formation NPHP4 to abnormalities although flagellar contrast IFT Therefore, in severe impaired in is greatly results IFT which on and loss, loss CEP290 NPHP4 of of case, effect effect any slight In altered section). very or above the regeneration (see impaired concentrations normal loading, ion for intraflagellar cargo that necessary IFT kinetics signaling reduced regeneration flagellar reflect slower The might IFT. observed on we effect 2011) al., et results in These of those machinery. with IFT is slightly consistent the generally only affect are this directly is not for velocity does NPHP4 and reason steady-state normal in the is reduced frequency Presently, IFT impaired. unclear. significantly are r ieyt eivle nmitiignra nrfaelrCa intraflagellar normal maintaining in involved be to likely are A eim(omnadLvn,1965). Levine, and (Gorman medium TAP Sllwe l,20)wsisre nothe into inserted was 2001) al., et (Silflow NPHP4 ewe h eutmt n ataioais,a tag acids), 3xHA amino a last (containing and penultimate NPHP4–HAC the encoding between construct a generate NPHP4 rmtepxApamdwssblndit the into subcloned clone was plasmid p3xHA the from with B1179 of with 79D8 ( of transformation by generated oiidwt . MKH mM 2.2 with offspring modified selected of transformation and with sequentially) 137c, and (CC124 IS- l A lnswr ucae rmCesnUniversity Clemson from purchased were clones pBluescript BAC into Institute. inserted All Genomics and 34B19 SK-. clone BAC II from cut were fragment nphp4-1 NPHP-4 , ihtepR78pamd(euh ta. 99.The 1989). al., et (Debuchy plasmid pARG7.8 the with ) DRC3 mt , DRC3 nphp4-1 , PP-A ( NPHP4-HAN . mt+ 2 , drc3 eeotie rmthe from obtained were ) NPHP4-HAC nphp4 el eegoni eimI(ae n rnc,1953) Granick, and (Sager I medium M in grown were Cells . a eeae yisrinlmtgnsso 1 ( H11 of mutagenesis insertional by generated was ) 2225-bp a , , a oet(argie l,20)o o(Williams no or 2008) al., et (Jauregui modest has idtp tan 3c( 137c strains wild-type Spe NPHP4-HAC , DRC3 drc3 rgetwr u rmBCcoe1L9containing 18L19 clone BAC from cut were fragment I el r bet sebefl-eghfael,the flagella, full-length assemble to able are cells , , DRC3 mt , NPHP4 nphp4-1 drc3 )wsmd ytasomto f7D with 79D8 of transformation by made was +) 2 BamH PO nphp4 Sma , olwdb w rse owl-yestrains wild-type to crosses two by followed , mt 4 DRC3 I– n . 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(Kindle, ta. 2002). al., transformation et For antibodies. anti-HA with immunofluorescence or by anti-NPHP4 screened with and containing microscopy plates selected medium randomly TAP were on Colonies zeomycin 1996). al., et (Stevens marker opeaencefaelraprtss PP–A el were cells NPHP4–HAC apparatuses, nucleoflagellar prepare To l rnfrain eepromdb h ls ed method beads glass the by performed were transformations All DRC3 6 el eec-rnfre ihthe with co-transformed were cells , 0m o oesiswr le oopst nso 25 a of ends opposite to glued were coverslips 1 No. 30-mm ora fCl cec 21)17 7442 doi:10.1242/jcs.155275 4714–4727 127, (2014) Science Cell of Journal , 20 the , m -epcabrfrosraino h el,22 cells, the of observation for chamber m-deep m )o h elcluewste lcdbtenthe between placed then was culture cell the of l) aph7 .rihrtiNPHP4 reinhardtii C. nphp4 0 eewsue steslcal akr(Berthold marker selectable the as used was gene n PP- el t10 at cells NPHP4-R and ˚ ,floe yfu ahswith washes four by followed C, a mlfe yPRwith PCR by amplified was a ble BamH tbln(ifo and (Silflow -tubulin 6 eea selectable a as gene objective. 4 2 –10 and I g 20 o 0mn The min. 10 for 5 ˚ )fr1 min. 15 for C) el/lwere cells/ml , 3m.An mm. 23 Sal 4 6 ,15mM sites, I 22-mm 6 b 75- F 1 -

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National the (to by GM060992 supported was research This Funding to helped and study this funded the data. and the prepared G.B.W. supervised interpret G.B.W. and and J.A. G.J.P experiments. manuscript. some with assisted initial K.F.L. the TESM. performed S.T. the experiments. of most characterization performed and designed J.A. contributions Author interests. competing no declare authors The interests Competing Johnson Coleman Eric the identifying thank for We MA) (VGN) spectrometry. (UMMS, Network mass Genetics Vermont for the for Facility to Facility Proteomics and Microscopy microscopy electron University Electron with the Core help to (UMMS) expert antibodies, School provided Medical generously who Massachusetts those of all to grateful are We Acknowledgements and 137c IFT of Observation 30 from obtained were Flagella point each at glutaraldehyde. (National values cells. 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To mixed western Bio-Rad). was Plus; for Stain flagella flagella (Silver stain whole silver prepare with visualized were bands ecie rvosy(riee l,21) yorpswr aeby made were plugin. Kymographs MultipleKymograph 2010). the al., with et ImageJ (Craige previously described oes n rnhCag UM,M)frciia discussion. critical for MA) (UMMS, Craige Branch providing and for models, MS) University, Mississippi, of 6 D-apeslto n nuae t75 at incubated and solution SDS-sample ora fCl cec 21)17 7442 doi:10.1242/jcs.155275 4714–4727 127, (2014) Science Cell of Journal nphp4 uuts5poendtbs Ei o,personal Hom, (Erik database protein 5 Augustus el at cells nphp4 uat .. ...adKEF curddt with data acquired K.E.F. and K.D.B. 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