Journal of Cell Science etn ,7ad9adMP ooaieaogthe length along Ghossoub ciliary Rania control colocalize and cilium MAP4 primary and the in 9 axoneme and 7 2, Septins Article Research irtbl tblt,ps-rnltoa oiiain and 2010). Spiliotis, modifications by (reviewed regulate post-translational and interactions microtubules, microtubule-vesicle stability, of subpopulations to microtubule and bind the Saarikangas colocalize and also at Septins with, by actin 2012). Cossart, (reviewed cortical and Mostowy with mitosis 2011; Barral, and during interphase furrow in actin cleavage cells with colocalize in septins to fibers cells, stress most In leads cytoskeleton. the with functions 2011; septin Barral, 2012). and Cossart, of and and Saarikangas Mostowy by furrow (reviewed cleavage perturbation defects the contractile cytokinesis to and the localize of assembly septins midbody, the the mammals, the to for In mother to scaffold and the ring. barrier a from mother and a mRNAs cell, the form and daughter proteins that between of In rings neck diffusion interlocking mammals. lateral the into to at bud of yeast daughter assemble regulators from septins important ranging yeast, organisms as in identified spirals. cytokinesis were and rings septins filaments, form Originally, into that GTPases assemble small that of family hetero-oligomers conserved highly a are Septins and cilium primary Introduction the of organization the in MAP4 and septins our together, of Taken words: regulation length. Key ciliary and regulate negatively functions of to the accessibility cells. appeared the it into in retinal controls where of activities and insights of axoneme members microtubule-based SEPT2 cilium-localized cilium the three new of of actin-based primary to the depletion partner provide cells, that localized binding the the epithelial a and also data in kidney is SEPT9 was septins sperm with in which and microtubules, observations of MAP4, the SEPT7 to to interact ciliogenesis. role with septins yeast, Similar inhibited the complex axoneme. and approaches budding the 1:1:1 investigated siRNA-based in of a hetero-oligomers We by length forms septins barriers cells. the SEPT2 form diffusion along epithelial that colocalize kidney form that found complex of and this and GTPases cilia cells, functions, (RPE) primary scaffolding of epithelial of pigmented in base family involved the conserved are and They flagellum evolutionarily microtubules. large, and cytoskeleton a are Septins Summary UK London, London, College Imperial Infection, and Bacteriology Molecular for Centre § MRC Microbiology, of ` Section address: *Present 10 9 8 7 6 5 4 3 2 1 Benmerah Alexandre and o:10.1242/jcs.111377 doi: 2583–2594 126, Science Cell of Journal 2013 March 20 Accepted eg Mostowy Serge hs uhr otiue qal oti work this to equally contributed authors These uhr o orsodne( correspondence for Authors NA S22,Prs France Germany Paris, Mainz, USC2020, University, INRA, Gutenberg France Johannes Paris, Zoology, U604, of INSERM, Institute Unite Biology, Pasteur, Matrix Institut and Cell of USA Department CA, Ho Stanford, U983, University, INSERM, Stanford Biology, of Department Universite France Paris, France UMR8104, Paris, CNRS Cochin, Insitut U1016, INSERM eateto oeua n ellrPyilg,Safr nvriy tnod A USA CA, Stanford, University, Stanford Physiology, Cellular and Molecular of Department 03 ulse yTeCmayo ilgssLtd Biologists of Company The by Published 2013. nmmas etn aebe ikdt cfodn functions scaffolding to linked been have septins mammals, In ai ecre,Sron ai Cite Paris Sorbonne Descartes, Paris ´ rmr iim etn,MP,Mcouue,Actin Microtubules, MAP4, Septins, cilium, Primary ptlNce-nat aae,Prs France Paris, Malades, Necker-Enfants ˆpital 7,8,9, e neatosBacte Interactions des ´ 1,2,3 [email protected] ,UeWolfrum Uwe *, iogHu Qicong , 1,2,3,5, ` ,§ 4 ; ainFailler Marion , [email protected] re-ells De ´ries-Cellules, 6 ,Isiu mgn,Prs France Paris, Imagine, Institut ´, oheSaunier Sophie , preetd ilgeCluar tIfcin ai,France Paris, Infection, et Cellulaire Biologie de ´partement 1,2,3,5 ) 3,5 ai-hitn Rouyez Marie-Christine , acl Cossart Pascale , h iir oprmn n h urudn lsamembrane plasma surrounding the against and compartment defense ciliary host the the in in participate 2010). involved al., bacteria also et also intracellular (Mostowy they pathogens are around that septins structures indicating Finally, cage-like complex Joo exocyst 1998). of in 2002; the of formation al., al., subunits annillin et et sec6/sec8 been the and Kinoshita (Hsu and 2000; have 2007), II al., al., partners et myosin et binding (Oegema non-muscle cytokinesis septin including Several identified 2012). and Mostowy 2011; Cossart, Barral, and of thereby, Saarikangas by morphogenesis by and (reviewed membrane neurons trafficking vesicular and, plasma mitosis, in the participate phosphatidylinositol-4,5-bisphosphate of sub-domains binding Septins with 2010). Spiliotis, associate by (reviewed is also 2008) 2009), al., et al., and al., Spiliotis et 2005; (Kremer et binds interactions septin-microtubule Holmfeldt regulate directly to by thought (reviewed which microtubules MAP4, bundles microtubules. MAP2/ cytoplasmic of polymerization/stability ubiquitously the regulates positively a of which family, MAP4, TAU (MAP) with protein interacts microtubule-associated directly expressed (SEPT2) 2 Septin etn raieltrlmmrn ifso aresbetween barriers diffusion membrane lateral organize Septins 7,8,9 .JmsNelson James W. , 1,2,3 ejmnSpitzbarth Benjamin , 4,10, ` ,§ 6 , 2583 Journal of Cell Science hhe l,21;Gri-ozl ta. 01.I MD cells, IMCD3 In 2011). al., 2010; et al., Garcia-Gonzalo et 2012; (Hu al., fibroblasts cilium et primary and the Chih cells of (IMCD3) base kidney the murine at in barrier diffusion a the forms to SEPT2 localize and complex cilium a primary form SEPT9 SEPT7, SEPT2, therefore cilium. and the Results in the length septins to controls of ciliary localized function positively the also regulates antagonize it is to that MAP4 negatively appears cilia. and and study of axoneme, 1:1:1 length axoneme, to the the a along and models of forms localizes ciliogenesis complex length SEPT2 used this cells entire that widely SEPT9, RPE1 the and most in SEPT7 with that the complex found of We one ciliogenesis. cells, [hTERT-RPE-1 epithelial (RPE1)] pigment linking retinal th thereby telomerase-immortalized investigated 2012), we cilium, al., primary et morphogenesis. al., Chih and et function 2010b; (Hu ciliary ciliogenesis al., to for septins et required also Kim is 2010; SEPT2 (Francis a cilium 2011). the al., with from to et proteins proposed agreement membrane been In of has in zone. exclusion membrane explain transition plasma is the the 2010b; at localization al., at retention et addition, barrier This Kim diffusion 2010; 2012). septin-based al., putative et al., (Hu et cilia Nelson, ring-shaped of Chih and a base form Hu the Septins at by 2011). structure (reviewed Marshall, filter complex and pore Ishikawa molecular 2011; nuclear the a to the similar form of cytoplasm the may role and and compartment zone cellular ciliary (Gilula the transition links the between The molecular onto to 1972). Y-shaped doublets docked by Satir, microtubule is membrane link ciliary to body the distally basal includes extends zone the and transition transition membrane, the where The by body, cilium. region basal primary encoded (reviewed the the the proteins near within the ciliopathies localized or all zone are as not genes if to these Many, by 2011). referred al., et generally with Hildebrandt obesity associated are and diseases polydactyly that genetic degeneration, retinal complex kidney, in polycystic identification mutated the genes by tissue cilium demonstrated of and primary clearly development The was type. during This cell homeostasis. pathways development the signaling during on key depending vertebrates controls adult in the cells in most and in present is cilia, Ishikawa the 2011). 2008; Rosenbaum, Marshall, to et the and back Pedersen by trains of (reviewed IFT body end basal of distal by transport balanced retrograde assembling is dynein-dependent transport the anterograde kinesin-based towards this axoneme; and axoneme import intraflagellar into select, trains) the an or to particles along thought (IFT requires termed components is ciliary that machinery transport and IFT machinery process The the (IFT). complex transport of conserved a assembly The is evolutionarily of body. a (ciliogenesis) bundle basal from a the assembled cilium is of called which centriole composed axoneme, modified are motility the cell Cilia termed in flagella). and microtubules, and cilia), (motile cilia in fluids (motile of motile involved movement and are the (primary al., cues cilia), that et environmental eukaryotes transducing (Kim and in are cilia sensing cilia structures 2005), and motile Flagella al., conserved and 2011). Nelson, et highly 2010) and Ihara Hu al., by 2005; (reviewed et al., 2010b) (Hu et cilium (Kissel flagellum primary sperm the in 2584 ofrhrcaatrz hs e ucin fspisi the in septins of functions new these characterize further To non-motile sensory of subtype specific a cilium, primary The ora fCl cec 2 (12) 126 Science Cell of Journal itiuino etn nhuman in septins of distribution e eo) ET,SP7adSP9clclzdwt AcTub with colocalized SEPT9 and (see cells SEPT7 ciliated of SEPT2, majority great the below), cell in the addition, throughout In filaments below). actin (see non- with fibers colocalized cytoplasmic in that as (and 1C) organized ciliated (Fig. were In septins cells) cells. apparently RPE1 RPE1 ciliated ciliated an in investigated in then present RPE1 ciliated is in 1B). SEPT2 SEPT2/SEPT7/SEPT9 (Fig. cells. of that blotting complex western show equimolar SDS-PAGE and results by 1A) (Fig. analyzed The GFP staining and silver septin with S-beads, using expressing by precipitated identify cells followed sequentially ciliated To antibodies from were (AcTub). lysates (Molla- cells, SEPT2–S-tag–GFP cilium tubulin these a 3–4 in acetylated a complexes formed as for identified cells was Under which stained the ciliogenesis. 2010), al., induce of et to Herman 80% hours conditions, 24 for these medium serum used low widely a minor and cells, a distribution RPE1 ciliogenesis. the for in in analyzed model characterize septins we further axoneme of cilium, To 2010). primary function the al., the et along at (Hu septins cells distributed the of also proportion is SEPT2 eut niaeta pcfccmlxo etn (SEPT2/ septins cells. of RPE the these in complex to cilium Altogether, primary specific localization artifact. the a to its fixation localized that SEPT7/SEPT9) that a indicate showing not results 2010), was al., cilium primary et Hu (Ha 2008; a marker colocalized below), see membrane also (SSTR3–GFP; 3 SEPT9–tomato ciliary type cells cells, receptor fixed somatostatin live the in In with cilium shown). primary the not in (data with AcTub cells with fused RPE1 colocalized in SEPT9_v1 also expressed S1). transiently cell and Fig. (SEPT9–tomato) RPE Tomato human material another (supplementary cells, ARPE19 Similar lines in cilium. primary obtained the were in results presence their indicating 1C), (Fig. nalclae P1cls(e eo)adol naminor a in only and below) (see cells RPE1 ciliated detected all not was axonemes in within septins of localization Because cells primary RPE1 mature in long cilia of axoneme the in accumulate Septins for the of obtained lines components cell tissues are were RPE different multiciliated in septins tubule cilia results Thus, bronchus primary of kidney shown). similar axoneme of not of SEPT9 2C); cilia (data (Fig. SEPT7 cilia retina, and cells primary the 2B) (Fig. epithelial in at cells the SEPT2 AcTub epithelial in to with be ciliary SEPT2 addition, colocalized represent to of which In remains daughter 2A), staining modifications. (Fig. staining the segments found this outer surrounding we photoreceptor of spots Moreover, significance in determined. the present but part). also bottom centriole 2A, was (Fig. cells adjacent SEPT2 the photoreceptor and of body basal centriole the the daughter cilium, connecting the in centrin3 of marker with colocalized a abundant SEPT2 cells. presynaptic nuclear was photoreceptor the outer of contains which terminals the SEPT2 layer, both plexiform outer in 2A, the retina and Fig. layer human human the in of various layer shown photoreceptor of As immunohistochemistry cilia tissues. of by axoneme analyzed the at further cilia septins of of axoneme presence the The at present are Septins h oaiaino h ET/ET/ET ope was complex SEPT2/SEPT7/SEPT9 the of localization The to transferred then and confluency to grown were cells RPE1 nvivo in . ne ta. 99 ebr tal., et Berbari 1999; al., et ¨ndel nvitro in nsitu in nsitu in n nclaof cilia in and nvitro in m o that rod m a then was cell Journal of Cell Science ET rd,adDP bu)t ti h uli aeso h ih r nagdveso ersnaiecla(oe ntemi mgs.Wiearrow White images). main the in (boxed S cilia representative SEPT2, of against 5 views antibodies enlarged bar: are green), Scale right (AcTub, field. the silver cilia same on for stain Panels the ( processed nuclei. to (B). in and the antibody SEPT2 cilia stain SDS-PAGE tubulin and to other by anti-acetylated SEPT7 (blue) separated DAPI SEPT9_v1, using and proteins to immunofluorescence (red), and antibodies for SEPT9 of (IP), processed mixture S-beads were a by hours, with followed 24 blotting GFP western or or (IgG) (A), antibody staining control with either immunoprecipitated ersnaieclaytf.Saebr 5 bar: Scale tuft. centriol ciliary the representative at centrin3 with SEPT2 indire of magnification colocalization 25 high partial top, layer. shows reveals bars: plexiform panel images Scale (IPL) Merged bottom retina cilium. inner segment. re The human connecting magnification and outer (INL). high the the and (OPL) layer The In along inner outer nuclear cells. (blue). and the the photoreceptor inner DAPI (BB) human in of and through pattern with body compartments (ONL) cryosections fibrous stained synaptic longitudinal outer a were in the the as (green) nuclei and of centrin3 localization and (IS) nuclei and (Ce), segment (red) the centriole SEPT2 inner in of adjacent the detected immunofluorescence the (OS), was and segment staining body outer weak basal the Additional (CC), in cilium localized connecting is cilia the SEPT2 of for axoneme marker the as to used localize Septins 2. Fig. cells. RPE1 of cilium primary the at complex SEPT2/SEPT7/SEPT9 A 1. Fig. uuelmn r niae L) ihrmgiiain frpeettv ii r hw ntergt n te ii r dniidwt ht arr white with identified Repre are (blue). cilia other DAPI and with right, stained the were on Nuclei shown green). are (2nd; cilia only representative antibodies of anti-rabbit magnifications secondary Higher 5 with (Lu). indicated or are (green) lumens SEPT9 tubule either and (red) AcTub against m .( m. C isescin rmteln fa3-ekftswr tie ihatbde gis cu rd n ET gen.Wiearw on oa to point arrows White (green). SEPT9 and (red) AcTub against antibodies with stained were fetus 33-week a of lung the from sections Tissue ) m m. m m. nvivo in . ( A nietimnfursec rpesann fSP2(e) eti sfr gen,wihwas which (green), 3 isoform centrin (red), SEPT2 of staining triple immunofluorescence Indirect ) m ;bto,1 bottom, m; m .( m. ( A B , B etn n A4rglt iir egh2585 length ciliary regulate MAP4 and Septins isescin rmauthmnkde eesandwt antibodies with stained were kidney human adult from sections Tissue ) P1clsepesn ET–-a–F eeetatdadproteins and extracted were SEPT2–S-tag–GFP expressing cells RPE1 ) C P1cls rw ncvrlp n eu-tre for serum-starved and coverslips on grown cells, RPE1 ) w.Saebar: Scale ows. el SEPT2 veals ,tebasal the e, P7or EPT7 indicate s sentative ct , Journal of Cell Science eovltd(etpnl)ad3 eosrciniae rgt hwdta ET tiigoelp ihATb cl as 5 bars: Scale AcTub. with overlaps where staining images) SEPT7 main that the in showed (boxed (right) cilium images representative reconstruction a of 3D views and enlarged panels) are (left right boxes the deconvoluted (white on ( cilium Panels staining. (red). each NPHP1 SEPT7 indicate of ( against arrows views respectively. and polyclonal enlarged acetylated-tubulin stainings, rabbit against are analyze a SSTR3 antibodies ( right and directly monoclonal and the (green) images). mouse and SEPT9 on of main hours of mix Panels the 48 a end shown. in using for are distal immunofluorescence (boxed serum-starved cilia the cilia (E) fusions, indicate anti- representative long (red) arrowheads an of and SEPT9–tomato and using (D) views and short obtained enlarged (green) Representative were are SSTR3–GFP microscopy. results right encoding epifluorescence Similar the plasmids (red). on with anti-SEPT9 Panels transfected and S2). transiently green) Fig. (AcTub; material tubulin (supplementary acetylated antibody against antibodies using immunofluorescence 4(4h r4 4 )huswr ye n xrsino ET,SP7adSP9wsaaye ywsenbotn ihteidctdatbde,expressi antibodies, indicated the with blotting western by analyzed was SEPT9 and SEPT7 SEPT2, of expression and lysed cells. were RPE1 hours in h) cilia (48 mature of long 48 of or axoneme h) the (24 to 24 localize Septins 3. Fig. serum low a in grown were cells ( hours RPE1 6 for different cilia. thereby, concentration and, of serum of lengths withdrawal after times different the of pool minor very septins. that a cellular of and represents total cells probably network not cilium non-ciliated the cytoplasmic and at ciliated overall septins was in agreement the similar appears in SEPT2/SEPT7/SEPT9 that septins result observations of a the the ciliogenesis, shown with As in expression upon hours. modified 48 grown or 3A, significantly 24 Fig. cells of for serum level RPE1 low in in expression or in serum, the of ciliogenesis Thomas examined presence by we during (reviewed Therefore, SEPT2/7/9 family 2010). factors al., transcription cilium et controlled RFX program of the transcription specific by state a to on owing ciliogenesis dependent was cilium (Hu cells primary maturation. septins of epithelial the incorporation IMCD3 the whether into in investigated we cilia 2010), al., primary et the of proportion 2586 c et eeaie etnlclzto ntepiayclu at cilium primary the in localization septin examined we Next, during upregulated is proteins ciliary some of expression The tblnwsas nlzdi h aecniin sacnrl ( control. a as conditions same the in analyzed also was -tubulin ora fCl cec 2 (12) 126 Science Cell of Journal , G 1 P1clssavdfr4 or eesandfraeyae-uui ATb re)adSP7(e) tcso mgswere images of Stacks (red). SEPT7 and green) (AcTub, acetylated-tubulin for stained were hours 48 for starved cells RPE1 ) m rmr iim i.3)and 3B) Fig. cilium; primary m B , C P1cls eu-tre o B r4 or C,wr rcse for processed were (C), hours 48 or (B) 6 for serum-starved cells, RPE1 ) ( A P1clsmitie ntepeec fsrm()o rw nlwsrmfor serum low in grown or (S) serum of presence the in maintained cells RPE1 ) rcs htpoal eed ntergaulaccumulation gradual their on depends ciliogenesis. probably during by that cilia These primary process shown). mature not in a accumulate (data septins not Note, fusion that observations indicate SEPT2–GFP Similar 3E). did results a cilia. (Fig. primary using arrowheads) it of made cilia tip were whereas 3E, the at primary (Fig. 3D), localize (Fig. to long SEPT9–tomato appear cilia of that primary length however, short the of 2D,E). decorated (Fig. base only population the long or cell absent and was stained the SEPT9–tomato short SEPT9, in endogenous of to detected mixture Similar were a that starvation, cilia cells serum primary RPE1 after live hours Forty- primary in eight SSTR3–GFP. and mature confirmed SEPT9–tomato co-expressed long, was transiently observation in Fig. This material accumulate cilia. (supplementary staining septins long SEPT7 Therefore, septin for 3B), S2A). strong Fig. obtained were had hours, (6 results hours) (48 cilia 2C; from cilia ‘young’ (Fig. absent primary short was SEPT9 of ‘mature’ Although axoneme 3). (Fig. the SEPT9 and AcTub 8hus( hours 48 F P1cls eu-tre o 4huswr rcse for processed were hours 24 for serum-starved cells, RPE1 ) , . c tbln(AcTub+ -tubulin 4 0 fspi-oiiecla e eo) Similar below). see cilia, septin-positive of 80% m rmr iim i.3) ie n tie for stained and fixed 3C), Fig. cilium; primary m c u;bu) otplcoa gis NPHP1 against polyclonal goat a blue), Tub; m m. D , E P1clswere cells RPE1 ) SEPT7 .Arrows ). h white the dby on Journal of Cell Science eed natnplmrzto Knsiae l,20) we 2002), al., filaments et cytoplasmic (Kinoshita on 2010). polymerization al., septins actin et of Pitaval on 2010; depends distribution al., et the ciliogenesis Bershteyn in Because 2010a; involved al., be et to (Kim recently shown were septins cytoskeleton cells, RPE1 with cilium. and in cytoplasm, the cilium the in that in microtubules filaments the appears actin with actin within it colocalize general mainly with except Therefore, the with 4A,b) localized 4Ac). not (Fig. (Fig. but mainly population 4A,a), (Fig. SEPT7 microtubule cytoplasm cells, the in arrows). RPE1 mitotic filaments S2C, the Fig. ciliated at material microtubules In (supplementary with midbody and not cortex or but cell spindle furrow, the cleavage at the actin at filamentous with mitosis, colocalized During SEPT7 S2B). Fig. AcTub-positive material cytoplasmic (supplementary with microtubules not but actin fibers) with In (stress colocalized shown). filaments SEPT7 not cells, data RPE1 and interphase with S3, non-ciliated made Fig. had were material observations (supplementary analysis SEPT9 similar SEPT9 detailed although a 4), and show (Fig. we SEPT7 SEPT7 We so of 1), SEPT2, 2010). Fig. and (see cells; Spiliotis, cytoskeleton distributions identical RPE1 actin by the in (reviewed septins, microtubules of types distribution cell the compared other cytoplasmic epithelial and stable (MDCK) kidney with cells canine and Madin–Darby types, in in cell microtubules cytoplasm non-dividing the throughout all cables almost actin with colocalize polymerization Septins cells actin RPE-1 of of cilium independent primary is the to septins of Localization hwda lotcmlt vra ewe cu and along cells. RPE1 AcTub all in accumulate cilia between mature septins long, overlap that of clearly axoneme indicating the complete 3G) images (Fig. Three- almost SEPT7 deconvoluted 3F). (Fig. an of markers axoneme showed other reconstruction the the the of no of dimensional or any compared little markers with detected we we colocalization AcTub, of with cilium, colocalized that SEPT9 ( Whereas primary body to basal the SEPT9 (AcTub), of of present regions distribution were septins other whether examine To at 3). 1, (Figs cilia mature h oyeiainsaeo otatlt fteactin-based the of contractility or state polymerization The etn ooaie ihATbwti h xnm flong of axoneme the within AcTub with colocalized Septins c tbln n rniinzn (NPHP1). zone transition and -tubulin) novddrcl ntesed tt oaiaino etn tthe at septins of localization state be steady cilium. to the appear material in not does directly (supplementary cytoskeleton involved actin-based unaffected the Thus, again the S3). Fig. in was septins of cilium staining whereas primary of increased, by their cortex inhibited and the shorter at addition was staining were II filaments primary myosin However, septins the previously cytoplasmic non-muscle in blebbistatin, When 2002). localization as 4B). SEPT7 (Fig. affect al., cilium 4B), not (Fig. did that et D structures aggregates cytochalasin (Kinoshita ring-like actin into described distribution with filaments the in septin colocalized of change dramatic localization cytoplasmic a using the of filaments in actin on resulted of D Disruption polymerization cytochalasin cilium. actin primary the of to septins role the tested ET–oaowr mgdb iecl fluorescence the along distributed live-cell uniformly was and cells by live SSTR3– in we cilia with in colocalized and imaged GFP trains, SEPT9–tomato 5A). SSTR3–GFP IFT (Fig. co-expressing were microscopy the IFT- cells in for involved RPE1 SEPT9–tomato rails are movements. septins the axonemal like not forms or whether axoneme examined the Since and stable, non-dynamic are septins cilium-associated Primary ET–F loecne(ros a oprdt htof that to primary-cilium-associated fluorescence compared Whereas (arrowheads). of was filaments septin rate (arrows) cytoplasmic 5B; recovery fluorescence (Fig. the SEPT2–GFP photobleaching fluorescence boxes), live-cell Following white by 5B–D). imaged (Fig. basal were the microscopy SEPT2–GFP identify and to pericentrin–RFP body, fluorescence co-expressing pool. cytoplasmic whether cells using the tested with RPE1 exchanged first axoneme dynamics the We within (FRAP). septins their photobleaching after investigated recovery we cilium, body 2008; basal Rosenbaum, and the 2011). Pedersen Marshall, at by et accumulate (review Ishikawa particles tip and IFT distal cilium the of the and/or cells organization within are punctate the fixed move distributions more of that These in length the shown). not the from observed data along different and as localized 5B–D, also arrows) (Fig. cilium SEPT2–GFP 5A, 2). (Fig. 1, cilia (Figs of length ofrhrepoeteognzto fspisi h primary the in septins of organization the explore further To etn n A4rglt iir egh2587 length ciliary regulate MAP4 and Septins ET-oiiecla rmtreidpnetexperiments independent ( three from cilia) SEPT7-positive n hntetdwt 5 with treated then the and and (b) ( microtubules (c). (a) cilium main fibers the in stress (boxed including cell images) same the views of enlarged regions are representative right of the on Panels (blue). filaments actin for stained (red), and SEPT7 a fixed against then antibodies and using hours immunofluorescence 24 for starved serum ht rospitt ii ntesm il.Saebars: Scale field. same the in images). cilia 5 main to the point in arrows enlarged (boxed White are cilia right representative the of on views Panels blue). fluorescent (actin; and phalloidin green) (AcTub; (red), tubulin SEPT7 acetylated against antibodies using immunofluorescence oto,wt h aefnlcnetaino MO for DMSO, 37 of at concentration minutes final 30 same the with control, does polymerization. cilium actin primary on the depend to not septins of Localization 4. Fig. n tbln( -tubulin 5 m .(a ih)Rslswr uniid(..pooto of proportion (i.e. quantified were Results right) (Far m. 0claprexperiments). per cilia 40 a B Tb re) n loecn hlodnt stain to phalloidin fluorescent and green), -Tub; P1clswr eu-tre o 4hours 24 for serum-starved were cells RPE1 ) ˚ ,te ie n nlzdby analyzed and fixed then C, m yohlsnD(yo)o,a a as or, (CytoD) D cytochalasin M ( A P1clswere cells RPE1 ) Journal of Cell Science htbece ein fSP2GPddntapa to The appear cilium. not primary did or the SEPT2–GFP 5C) (Fig. of of distal domain regions the 5D) photobleached in (Fig. photobleached proximal was SEPT2–GFP distinct with each with exchange associated to appear not represent being other. do that septins data pools to cilium-associated dynamic These different addition and SEPT9– S5). cytoplasmic in the Fig. structures, than using that material more obtained to (supplementary indicate over were extended fusion was results slowly tomato time Similar did recovered recovery cilium minutes. the 50 septins primary when the of even in not, fluorescence pool SEPT2–GFP minutes, cytoplasmic 4 the 2 bars: Scale of photobleaching. before arrows cilia White the period. of indicated position the the of part for recovery to distal and analyzed point photobleached the was were B), fluorescence cilium min; the GFP of (27 the (D) filament part whole proximal SEPT2 the the cytoplasmic or was including a (C) (B) images) or main filaments min) the cytoplasmic (0 in in cilium (boxed or Regions The (B–D) FRAP. microscopy. cilium by confocal the analyzed live-cell at by SEPT2 analyzed of serum- and dynamic then hours fusions, 48 (red) for the pericentrin–RFP starved of and position (green) every the SEPT2–GFP image indicate for (one arrows shown White is right). ( cilium to cilium. long left a from in example seconds; SEPT9 An 5 microscopy. of epifluorescence behavior live-cell the for by of serum-starved analyzed then and fusions, hours (red) cilium. 48 SEPT9–tomato primary and the (green) at GFP pool dynamic stable-non a ( form Septins 5. Fig. 2588 A P1clswr rninl rnfce ihpamd oigfrSSTR3– for coding plasmids with transfected transiently were cells RPE1 ) ots hte etn r yai ihnpiaycilium, primary within dynamic are septins whether test To B–D ora fCl cec 2 (12) 126 Science Cell of Journal P1clswr rninl rnfce ihpamd coding plasmids with transfected transiently were cells RPE1 ) m m. ET/ET ope ly oei iignss n that and cilia. ciliogenesis, SEPT2/ long in form the to role that required a is indicate SEPT9 plays results These complex 6D). SEPT7/SEPT9 (Fig. cells treated 3.0 el eesotrta ncnrlcls(1.69 cells control in SEPT9-depleted than in cilia shorter primary were (92.9 However, cells cilia 6C). of Fig. formation control; the of on effects limited in resulted oto;Fg BC n h rmr ii htfre were formed that cilia (1.81 primary 2.71 cells versus control-siRNA-treated the in (49.9 those and than shorter was ciliogenesis 6B,C) cilia Fig. of inhibited with Depletion control; cells significantly ciliogenesis). of (normalized percentage SEPT7 The controls 2010). to al., normalized et al., et of Hu based (Molla-Herman previously quantified 2010; proportion described were as the cilium (see staining, primary AcTub and of on length complexes immunofluorescence, and by cells septin ciliated cells RPE1 of in stability overall by of the function confirmed Discussion). key in the were identify results SEPT7 and 6B), These (Fig. Discussion). immunofluorescence (see observed a easrcua opnn fteaxoneme. the and of dynamic component not structural are a septins results be that the indicate may Nevertheless, cilia to cilia. primary unable mature those were in in we dynamics weak, in very septin was SEPT2–GFP analyze cilia of primary fluorescence immature the short, Since fluorescence. recover ncloeei ete sdpeiul hrceie siRNA characterized of previously expression used knockdown to then sequences we ciliogenesis for in necessary these is network Thus septin S4D,E). dynamic a ciliogenesis. 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RPE Fig. another 3, cells, 1, the ARPE19 (Figs in observed cells were in SEPT2/SEPT7/SEPT9 the RPE1 of of distributions distributions quiescent axoneme overlapping the of overlapping at cilium prominently primary and had cells, RPE1 isoforms of cytoplasm septin these etn rmsal yolsi irtbls(rmre al., competition similar et a whether (Kremer test To microtubules 2008). al., et cytoplasmic Spiliotis 2005; not stable cilia. of is from axoneme septins MAP4 the to that septins of indicate versus recruitment (84.0 the data cells for control These required for that respectively). 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(boxed views cilia enlarged representative are of right the DAPI on with Panels stained (blue). were nuclei the (AcTub; and tubulin blue), acetylated and (red) for SEPT7 antibodies with immunofluorescence processed for and fixed plasmids, or GFP-MAP4m SSTR3-GFP with transfected transiently experiment, he needn xeiet ( from experiments conditions independent both three in quantified was SEPT7-positive cilia of proportion main The the images). of in views (boxed enlarged cilia are representative right the (red). on SEPT7 Panels and green) (AcTub, were acetylated tubulin 8) against Fig. antibodies in with (as stained control a as siRNA (siLUC) targeting luciferase or siMAP4(1) i.9 A4cmee ihspisa the at septins with axoneme. competes MAP4 9. Fig. ( A P P1clstetdwith treated cells RPE1 ) . .) ( 0.1). m m. B P1clswere cells RPE1 ) n 5 0i each in 30 Journal of Cell Science ecie Kee ta. 05 olye l,20;Sli tal., et Sellin 2009; al., of et previously knockdown Tooley efficient As 2005; 2011), al., approach. et siRNA-based (Kremer a described using septins depleted membrane membrane. of ciliary movement the the in prevent proteins to cilium appear not whole does axoneme S9B,B the (supplementary membrane Fig. ciliary of the material within low S9A,A diffuse with photobleaching also can 2012), Fig. GFP RPE-1 al., after material et of recovery Chih cilia (supplementary 2010; any the al., indicate et in if results (Hu similar cells Our IMCD3 are cells. and dynamics RPE1 ciliary of well-characterized SSTR3–GFP investigated cilia a that to in SSTR3–GFP, used IFT protein, also the was membrane of FRAP of Finally, dynamics cell. the with the exception exchange of limited rest very and the the or membrane low previous ciliary have with the compartments support axonemal molecules, of also signaling that, and properties They components dynamic indicating septins. the ciliary observations in and differences cytoplasmic indicate data These 2592 h feto A4dpeino iir eghcudbe could length ciliary on depletion MAP4 shorter stabilizes of of in Part resulted effect S10). which and Fig. the material depletion 2011), (supplementary MAP4 taxol, al., cilia of et MAP4-depleted effect (Sharma of the length ciliary reversed cells cells concentration decrease RPE1 RPE1 and in MAP4-depleted microtubules moderate of MAP4 treatment depleted with expected, we similar As when 2011), 8). al., observed (Fig. et we (Sharma cilia what of longer to amount in the Holmfeldt results increases tubulin, 2002; nocodazole, free and al., with microtubules treatment et depolymerizes and moderate which Holmfeldt Interestingly, al., 1999; 2007). microtubules et al., al., et (Nguyen et tubulin stabilizes microtubule-binding Nguyen polymerized 1997; of the MAP4m) proportion microtubule the containing increases (including MAP4 or of wild-type in of of domain increase overexpression regulator forms whereas an tubulin, in mutant free human positive results of in pool depletion MAP4 the and MAP4 well-established S6). 7), and (Fig. Fig. polymerization, a cells material RPE1 (supplementary is in axoneme fibroblasts cilium the primary Spiliotis along primary AcTub 2005; the with septins al., colocalized of MAP4 of et 2008). functions (Kremer al., the et microtubules regulates cytoplasmic the which with of MAP4, elongation examined we the of in assembly role cells. the RPE1 crucial in conclusion, a cilium In of primary plays septins 2011). elongation within filaments al., the position septin et in terminal (Kim its role of crucial oligomers because data a recent filaments plays with septin agreement of SEPT9 in elongation and that is 2012), proper SEPT9 showing al., the of et for importance Chih The required 2010; cilia. is specifically al., for is et previously SEPT9 complex (Hu shown that cells as These SEPT2/SEPT7/SEPT9 IMCD3 cells, in shorter. RPE1 on SEPT2 the in effect were ciliogenesis no that for cilia or required al., little indicate primary et in Estey the resulted results 2009; and al., although 2011), et al., SiRNA- ciliogenesis, Tooley et also control. Sellin (see those 2010; the not SEPT7 but or in levels SEPT2 SEPT9 than of affected SEPT9 and shorter of depletion SEPT2 in targeted significantly complex, inhibited were the was cells of ciliogenesis members conditions, , these two Under other SEPT9. the of loss 0 fcls n h rmr ii nteremaining the in cilia primary the and cells, of 50% oivsiaetefntoso etn nRE ii,we cilia, RPE1 in septins of functions the investigate To ofrhrivsiaeterl fspisi h rmr cilium, primary the in septins of role the investigate further To ora fCl cec 2 (12) 126 Science Cell of Journal 9 .Tu h rsneo etn ln the along septins of presence the Thus ). 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Agence Defense to [grant R09087KS Q.H.]; of number Fellowship to Department Predoctoral a BC083077 Program [grant Health W.J.N.]; Research of to Institutes Cancer National GM35527 the by number supported was work This Funding manuscript. the corrected coauthors the the wrote all A.B. and manuscript; W.J.N. the R.G., experiments; W.J.N. the experiments; all the analyzed analyzed of A.B. tissues most and experiments; designed and reagents, the A.B. of and provided W.J.N. some performed Q.H., analyzed R.G., P.C. and B.S. designed and and expertise and S.S. samples, U.W., M.C.R. S.M., M.F., experiments; Q.H., R.G., contributions Author sharing with for Pereira G. Toure help help; A. discussions. and their results; for his new Institute Cellulaire’ important Cochin ‘Imagerie the for the of help from facility her providing people Henneveu for for immunohistochemistry; Legendre with Flora collaborators Anthony transfections; generous and immunofluorescence their reagents; all important thank authors The Acknowledgements nuclei) of The cilia/number staining. of AcTub (number the in to by staining) equal detected was (DAPI cilia cells nuclei primary ciliated of of of number number percentage the the and counted quantified image we were the each cells, cilium determine To primary ciliated ImageJ. of of and images length percentage microscope the fluorescence and using measured cells and ciliated of percentage The analysis Statistical iue nbokn ufradicbtdwt h ebaefr2hus lt were Blots hours. were 2 antibodies for membrane SEPT2 the and with incubated SEPT7 and SEPT9_v1, buffer BSA). blocking in 2% diluted 20, Tween 0.1% DTT, %N4,01M(NH M 0.1 NP40, 1% imager. infrared Li-COR a in scanned and antibodies IRDye800-conjugated and Alexa-Fluor-680- secondary with incubated subsequently Sga]fr3 iue t4 at minutes 30 for (Sigma)] ehoois.Imnbotn a efre sn h niae primary indicated the using ECL the performed using revealed transfer was and fluoride antibodies (Life system Immunoblotting polyvinylidene electrophoresis NuPage onto the technologies). using transferred Healthcare) GE and (PVDF, membranes SDS-PAGE by separated o muoltig el eelsdi yi ufr[.2MTi-C H7.5, pH Tris-HCl M [0.02 buffer lysis in lysed were cells immunoblotting, For etn n A4rglt iir egh2593 length ciliary regulate MAP4 and Septins ˚ nbokn ufr(0m rsHl H68 5 MNC,1mM 1 NaCl, mM 150 6.8, pH Tris-HCl, mM (10 buffer blocking in C 4 ) 2 SO 4 0 lcrl 0m rtaeihbtrcocktail inhibitor protease mM 10 glycerol, 10% , ˚ .Atrcnrfgto,cerdlstswere lysates cleared centrifugation, After C. ´ iu RG] Re [R.G.]; rieur + eeto i G Healthcare). (GE Kit Detection t ´ tssadFshrstsswr sdfor used were tests Fischer’s and -tests n .Sasyfrstimulating for Spassky N. and ´ inied France, de ile gion ` ed aRecherche la de re g SEPT2–S- . 6 100. Journal of Cell Science u .adNlo,W J. W. Nelson, and Q. Hu, M. Gullberg, and E. M. Sellin, P., Holmfeldt, M. Gullberg, and S. Stenmark, P., Holmfeldt, M. Gullberg, and G. Brattsand, P., Holmfeldt, s,S . aua .D,Rt,R,Flti .L,Hue,J n celr .H. R. Scheller, and J. Heuser, L., D. Foletti, R., Roth, D., C. Hazuka, C., S. Hsu, N. Katsanis, and T. Benzing, F., Hildebrandt, acaGnao .R,Cri,K . ieo-iur .S,Rmsai . Otto, G., Ramaswami, S., M. Sirerol-Piquer, C., K. Corbit, R., F. Garcia-Gonzalo, o,E,Sra .C n rml,W S. W. Trimble, and C. M. F. Surka, W. E., Marshall, Joo, and 3rd R., J. Yates, J., Thompson, H., Ishikawa, F. W. Marshall, and H. Ishikawa, iua .B n ai,P. Satir, Ha and B. N. Gilula, I. Mellman, and B. Lo, J., Sfakianos, S., S. Francis, i,S . hno . ak .J,O,E . hs,S,Ga,R . ei,R A., R. Lewis, S., R. Gray, S., Ghosh, C., E. Oh, J., T. Park, A., Shindo, K., Aza- S. T., Kim, Ideker, K., Lee, K., Ono, E., Suyama, S., Heynen-Genel, E., J. Lee, J., Kim, Goto, A., Kitano, A., Tanigaki, H., Tanaka, S., Yamada, A., Kinoshita, M., Ihara, oUW] eoie nPCfrrlaeatr6months. 6 after release for PMC in 241955 Deposited number U.W.]. agreement Seventh to [grant Community’s FP7/2009 European number Programme ‘SYSCILIA’ Framework [grant and Award S.M.]; Grant Career to a 233348 Advanced S.M.]; Research Council to Trust Research WT097411MA European number Wellcome [grant a Fellowship Development [M.F.]; (CORDDIM) majeur se,M . iCaoOier,C,Fos,C . eie .T n rml,W S. W. Trimble, and T. M. Bejide, D., C. Froese, C., Ciano-Oliveira, Di P., M. E. Estey, Mandelkow, and M. E. Mandelkow, U., Preuss, A., Ebneth, G., Drewes, u . iekvc . i,H,Sot .P,Ncuy .V,Siits .T and T. E. Spiliotis, V., M. Nachury, P., M. Scott, H., Jin, L., Milenkovic, Q., Hu, hh . i,P,Cin . hlui . oue,L . as .E,Snoa,W. Sandoval, C. E., P. J. 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