RESEARCH ARTICLE

LRRC45 contributes to early steps of axoneme extensionBahtiyar Kurtulmus1,2, Cheng Yuan1,2, Jakob Schuy1, Annett Neuner3, Shoji Hata3, Georgios Kalamakis4,Ana Martin-Villalba4 and Gislene Pereira1,2,*

ABSTRACTCilia perform essential signalling functions during development andtissue homeostasis. A keyevent in ciliogenesis occurs when the distalappendages of the mother centriole form a platform that docks ciliaryvesicles and removes CP110-Cep97 inhibitory complexes. Here,we analysed the role of LRRC45 in appendage formation andciliogenesis. We show that the core appendage proteins Cep83 andSCLT1 recruit LRRC45 to the mother centriole. Once there, LRRC45recruits the keratin-binding protein FBF1. The association of LRRC45with the basal body of primary and motile cilia in both differentiatedand stem cells reveals a broad function in ciliogenesis. In contrast tothe appendage components Cep164 and Cep123, LRRC45 was notessential for either docking of early ciliary vesicles or for removal ofCP110. Rather, LRRC45 promotes cilia biogenesis in CP110-uncapped centrioles by organising centriolar satellites, establishingthe transition zone and promoting the docking of Rab8 GTPase-positive vesicles. We propose that, instead of acting solely as aplatform to recruit early vesicles, centriole appendages form discretescaffolds of cooperating proteins that execute specific functions thatpromote the initial steps of ciliogenesis.

KEY WORDS: LRRC45, Centrosome, Cilia, Distal Appendages,Mother centriole

INTRODUCTIONThe primary cilium is a microtubule-based organelle that protrudesfrom the surface of several cell types in the human body. It hasmechanosensory and signalling functions as a result of itsassociation with receptors of important signalling pathways, suchas hedgehog (Shh) andWnt (Goetz and Anderson, 2010). Defects inprimary cilia formation or function are the underlying cause ofsevere genetic diseases, collectively named ciliopathies, in whichembryonic development and/or functioning of multiple organs inthe human body can be affected (Reiter and Leroux, 2017).The primary cilium emerges from the mother centriole that

converges into the basal body of the cilium. Each centrosome iscomposed of two centrioles, cylindrical structures consisting of anine-fold array of triplet microtubules and pericentriolar material(PCM). The two centrioles of a centrosome are different in functionand composition. The mother, but not the daughter centriole,

associates with an array of proteins, named appendage proteins,which give rise to the cilium upon cell cycle exit (Nigg and Stearns,2011).

Ciliogenesis is a multi-step process that requires the concertedaction of several components, including mother centriole-associatedproteins and the vesicular transport machinery, which, together, areimportant for the establishment and maintenance of the ciliarycompartment (Sánchez and Dynlacht, 2016). At early stages ofciliogenesis, the mother centriole associates with small ciliaryvesicles, most likely derived from the Golgi (Das and Guo, 2011;Qin, 2012). After docking to the mother centriole, small vesicles arefused, thereby forming a large ciliary vesicle that caps this centriole(Sánchez and Dynlacht, 2016). The fusion process depends on themembrane-shaping proteins EHD1 and EHD3, as well as theSNARE component SNAP-29 (Lu et al., 2015). Ciliary membraneestablishment also requires components of the Rab-GTPasecascade, which includes the GTPase Rab11, the small RabGTPase Rab8 and the Rab8 activator Rabin8 (Knodler et al.,2010; Nachury et al., 2007; Yoshimura et al., 2007). Rabin8 andRab8 accumulate at the centrosomes shortly after induction ofciliogenesis, implying a local activation of Rab8 to promote ciliaryvesicle formation (Westlake et al., 2011). Alongside the process ofciliary vesicle establishment, the CP110-Cep97 complex is removedfrom the mother centriole by targeted protein degradation (Spektoret al., 2007). Removal of CP110-Cep97 allows the extension ofmicrotubules to form the ciliary axoneme and establishment of theciliary transition zone (Kobayashi et al., 2011; Yadav et al., 2016).The transition zone serves as a diffusion barrier at the base of thecilium (Reiter et al., 2012). The intraflagellar transport (IFT)machinery also controls cilia formation and extension (Follit et al.,2006; Jurczyk et al., 2004; Pazour et al., 2002, 2000; Pedersenand Rosenbaum, 2008). In addition, large cytoplasmic proteincomplexes, so-called centriolar satellites, are involved in thetransport of building blocks to the basal body (Craige et al., 2010;Garcia-Gonzalo et al., 2011; Hori et al., 2014; Jin et al., 2010;Klinger et al., 2013; Kurtulmus et al., 2016).

Distal appendage proteins at the mother centriole are key for theinitial steps of cilia biogenesis. Distal appendage componentsinclude the C2-calcium-dependent domain protein C2CD3, Cep83(also known as CCDC41), the sodium channel and clathrin linkerprotein 1 (SCLT1), Cep123 (also known as Cep89), Fas bindingfactor 1 (FBF1) and Cep164 (Graser et al., 2007; Joo et al., 2013;Schmidt et al., 2012; Sillibourne et al., 2013; Tanos et al., 2013;Weiet al., 2013; Ye et al., 2014). The protein C2CD3 positively controlscentriole length (Thauvin-Robinet et al., 2014). In the absence ofC2CD3, centrioles lack Cep83 and, consequently, all additionaldistal components, given that Cep83 is necessary for binding ofCep123, SCLT1, Cep164 and FBF1 to the mother centriole (Tanoset al., 2013; Ye et al., 2014). Upon depletion of distal components,including Cep164, Cep123 and Cep83, the initial docking of smallciliary vesicles at the mother centriole does not occur. In addition,the inhibitory CP110-Cep97 complex is not displaced from theReceived 6 August 2018; Accepted 7 August 2018

1Centre for Organismal Studies (COS), University of Heidelberg, 69120 Heidelberg,Germany. 2German Cancer Research Centre (DKFZ), DKFZ-ZMBH Alliance,Molecular Biology of Centrosomes and Cilia Group, 69120 Heidelberg, Germany.3Centre for Cell and Molecular Biology (ZMBH), DKFZ-ZMBHAlliance, University ofHeidelberg, 69120 Heidelberg, Germany. 4German Cancer Research Centre(DKFZ), DKFZ-ZMBH Alliance, Division of Molecular Neurobiology, 69120Heidelberg, Germany.

*Author for correspondence (gislene.pereira@cos.uni-heidelberg.de)

G.P., 0000-0002-6233-8352

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mother centriole and axoneme extension is blocked. Distalappendages interact with components of the vesicular transportmachinery, including Rab8, as well as with protein kinases andphosphatases that influence CP110-Cep97 recruitment (Bielaset al., 2009; Cajanek and Nigg, 2014; Goetz et al., 2012;Humbert et al., 2012; Kuhns et al., 2013; Oda et al., 2014;Schmidt et al., 2012; Tanos et al., 2013; Xu et al., 2016; Ye et al.,2014). Therefore, the current model is that distal appendages areindispensable for vesicle docking at the mother centriole and allsubsequent steps thereafter.Despite the importance of appendage proteins for cilia

biogenesis, it is not fully understood how appendages are formedand how they regulate the initial steps of ciliogenesis. Here, weinvestigated the function of the leucine-rich repeat protein,LRRC45, in cilia formation. LRRC45 was reported to be requiredfor centrosome cohesion at the proximal end of centrioles, as part ofthe linker that holds duplicated centrosomes together (He et al.,2013). LRRC45 is recruited to the proximal end of the centrioles bythe protein C-Nap1 (also known as Cep250; He et al., 2013). Inaddition, a pool of LRRC45 was shown to associate with mothercentriole appendages (He et al., 2013), implying a direct function inciliation. We now show that LRRC45 associates with the distalappendages of the mother centriole in a Cep83- and SCLT1-dependent manner. LRRC45 was required for FBF1 but not Cep164or Cep123 centriolar localisation. Depletion of LRRC45 impairedciliogenesis at the level of axoneme extension independently ofC-Nap1. Our data indicate that LRRC45 is required for theestablishment of the transition zone and recruitment of Rab8 andsatellites to centrosomes but not for removal of the CP110-Cep97complex or ciliary vesicle docking at the mother centriole. Wepropose that distal appendage proteins cooperate to control ciliaryvesicle docking and axoneme extension at early stages ofciliogenesis.

RESULTSLRRC45 contributes to cilia formation independently ofC-Nap1LRRC45 was shown to associate with the basal body of primarycilia in retina pigment epithelial (RPE1) cells (Fig. 1A) (He et al.,2013). We also observed that LRRC45 associates with the basalbody of ciliated murine fibroblasts, neural stem cells and motile ciliaof ependymal cells (Fig. 1A,B). This indicates that LRRC45 mayhave a function in promoting ciliogenesis in several cell types,including cells with motile cilia.To understand the function of LRRC45 in ciliogenesis, we

depleted LRRC45 using three independent small-interfering(si)RNAs and a pool of 4 siRNAs. Loss of LRRC45 led to areduction of cilia formation in serum-starved RPE1 cells (Fig. 1C,Dand Fig. S1A-D). This defective ciliogenesis was not due toinefficient cell cycle arrest, as the majority of control and LRRC45-depleted cells arrested in the G1/G0 phase (Fig. S1D). In addition,primary cilia in LRRC45 depleted cells were shorter in comparisonto control depleted cells (Fig. 1E), indicating that LRRC45 isrequired for cilia formation and elongation. The cilia-loss phenotypewas specific for LRRC45, as it could be rescued by the expression ofmurine Lrrc45 (Fig. S1E-G). However, wewere unable to rescue thestrong cilia-loss phenotype obtained with the published Lrrc45siRNA sequence (siLRRC45-1, Fig. S1G) (He et al., 2013). Thissuggests that the LRRC45-1 siRNA has an off-target effect thatexacerbates the cilia-loss phenotype.LRRC45 associates with the proximal end of the mother and

daughter centrioles independently of the protein C-Nap1 (encoded

by CEP250) (He et al., 2013). In addition, a fraction of LRRC45associated with the mother centriole independently of C-Nap1(Fig. 1F). Because of this dual localisation, we asked which fractionof LRRC45 contributes to cilia formation. For this, we usedCEP250 knockout (KO) cells (Panic et al., 2015). These cells canform cilia similarly to WT RPE1 cells (Fig. 1G). Depletion ofLRRC45 in RPE1CEP250KO cells caused the same degree of cilialoss as observed in WT RPE1 cells (Fig. 1G). We concluded thatLRRC45 regulates ciliogenesis independently of its function at thecentrosomal linker.

Recently, a role for the daughter centriole has been implicated inciliogenesis due to its proximity to the mother centriole (Loukilet al., 2017). We therefore asked whether lack of cilia upon LRRC45depletion in WT and CEP250 KO RPE1 cells correlated with thedistance between both centrioles (Fig. 2). Under serum starvation,mother and daughter centrioles were significantly more separated inCEP250 KO in comparison to WT RPE1 cells (Fig. 2A). Yet,CEP250 KO cells were able to form cilia even when mother anddaughter centrioles were more than 2 µm apart (Fig. 2A-D). Thisindicated that the physical proximity of daughter and mothercentriole is not required for ciliogenesis. There was no increase inthe distance between mother and daughter centrioles irrespective ofwhether these cells had a cilium or not in either cycling (Fig. 2E) orserum-starved RPE1 cells lacking LRRC45 (Fig. 2F). Together,these data indicate that defective ciliation upon LRRC45knockdown is most likely unrelated to the proximity of motherand daughter centrioles.

Cep83 and SCLT1 direct LRRC45 to distal appendagesOur data indicate that the mother centriole pool of LRRC45contributes to ciliogenesis. Using specific anti-LRRC45 antibodies(Fig. S2), we determined the subcellular localisation of LRRC45 atthe mother centriole by super-resolution microscopy (Fig. 3).Pairwise co-localisation analysis between LRRC45 and distal(Cep123, SCLT1, Cep83 and Cep164) or sub-distal (ODF2)components by stimulated emission depletion (STED) microscopyshows that LRRC45 appeared as dotted ring-like structures aroundcentrioles (Fig. 3A-E, top views). Lateral images of the centriolerevealed that LRRC45 did not co-localisewith ODF2 (Fig. 3A). TheLRRC45 signal mostly mapped with Cep83 (Fig. 3B) and partlywith Cep164 or SCLT1 (Fig. 3C). Interestingly, Cep123 could beresolved in some cells as one or two rings (Fig. 3D, lower panel). Incells with two rings, LRRC45 localised between the two rings(Fig. 3D), whereas in cells with one Cep123 ring, LRRC45 locatedmostly proximal to it (i.e. between Cep123 and sub-distalappendages). Moreover, by using three dimensional-structuredillumination microscopy (3D-SIM), we found out that LRRC45 atthe mother centriole forms rings of 256-272 nm in diameter(Fig. 3F). The size of LRRC45 rings was significantly smaller thanthe one formed by the distal appendage protein Cep164 (350±36 nm) but similar to the rings of the distal component Cep123(288±49 nm) or the sub-distal appendage protein ODF2 (260±32 nm) (Fig. 3F). These data indicate that LRRC45 associates withdistal appendage components.

Previously, a hierarchical network of distal appendage proteinswas unravelled by siRNA depletion experiments (Tanos et al.,2013). To position LRRC45 within this network, we firstinvestigated its localisation upon depletion of appendagecomponents. Cep83 targets the appendage proteins Cep123,Cep164, FBF1 and SCLT1 to the mother centriole (Fig. S3).Centriole association of LRRC45 was significantly decreased upondepletion of Cep83 (Fig. 4A,B) or SCLT1 (Fig. 4C,D), but remained

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Fig. 1. LRRC45 localises to the basal body of ciliated cells and is required for cilia formation. (A) Localisation of LRRC45 (green) with distal appendageprotein Cep164 (red) at the basal body in ciliated RPE1 cells and adult NSCs; polyglutamylated (pGlu) tubulin and γ-tubulin (grey) serve as markers for cilium andbasal body, respectively (top panels). LRRC45 (green) localises to the basal body in ciliated NIH-3T3 cells (bottom panel). ARL13B and γ-tubulin (red) serve asmarkers for cilium and basal body, respectively. Scale bars: 10 µm and 2 µm (insets). (B) LRRC45 (green) co-localisation with Cep164 (red) in multi-ciliatedependymal cells lining the ventricle of adult mouse brain. Poly-glutamylated (pGlu) tubulin and γ-tubulin (grey) serve as markers for cilium and basal body,respectively. Magnifications of the centrosome area or indicated region in B are shown to the right. Scale bars: 20 µm and 5 µm (insets). (C) Representativeimages of ciliated and non-ciliated RPE1 cells treated with control or LRRC45 siRNAs and serum starved for 48 h. ARL13B (green) served as a ciliary membranemarker, polyglutamylated tubulin (pGlu tub) and γ-tubulin (red) served as markers for ciliary axoneme and basal body, respectively. Scale bars: 10 µm and 2 µm.(D) Quantification of ciliation in control and LRRC45-depleted cells after indicated times of serum starvation. The bar graph indicates the average from threeindependent experiments; numbers in the bars represent total number of cells analysed for each condition. siLRRC45 indicates the mean values obtained forciliated cells using siLRRC45-pool, siLRRC45-2 and siLRRC45-3. (E) Quantification of ciliary length upon treatment with respective siRNA oligos in C. Bar graphrepresents average of percentage of cells with indicated ciliary lengths measured upon control (blue, n=310) or LRRC45 (red, n=877) depletion in threeindependent experiments. siLRRC45 represents the mean values of ciliary length measured using siLRRC45 pool, siLRRC45-2, and siLRRC45-3. (F) LRRC45(red) localisation in WT and CEP250−/− RPE1 cells. Cep164 (green) and γ-tubulin (grey) serve as markers for mother centriole and centrosomes, respectively.The arrowhead indicates LRRC45 co-localising with Cep164, while the two asterisks mark LRRC45 at the proximal ends of centrioles in CEP250+/+ cells. Scalebars: 10 µm and 1 µm. (G) Bar graph shows percentage of ciliated cells after 48 h of serum starvation in CEP250+/+ and CEP250−/− RPE1 cells (clone 7 and 17)upon treatment with control and LRRC45 siRNAs. siLRRC45 represents the mean value in ciliated cells using both siLRRC45-2 and siLRRC45-3. CEP250+/+:siControl n=339; siLRRC45, n=802; CEP250−/− cl.7: siControl, n=325; siLRRC45, n=793; CEP250−/− cl.17: siControl, n=300; siLRRC45, n=838. siControl-treated CEP250+/+ cells are normalised to 100%. DAPI (blue) stains the nuclei in A-C and F. Error bars represent s.d. *P<0.05, **P<0.01, ***P<0.001, n.s., notsignificant (Student’s t-test).

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unaffected upon depletion of Cep123, Cep164, FBF1 or ODF2(Fig. S4A-D). This indicates that LRRC45 is mainly targeted todistal appendages via Cep83 and SCLT1. Upon LRRC45 depletion,the levels of FBF1 at mother centrioles were significantly decreased(Fig. 4E,F), whereas the levels of ODF2, Cep123, SCLT1 andCep164 did not change in comparison to control-depleted cells(Fig. S5A-D). Together, our data are consistent with the modelwhereby LRRC45 is positioned between Cep83, SCLT1 and FBF1in the organisational scheme of distal appendage components(Fig. 4G).We next employed the yeast two-hybrid system to determine the

interaction network of distal appendage proteins, including LRRC45.For this, we used a combination of full-length and truncatedconstructs to identify their minimal interaction domains (Fig. 5A).We reasoned that interactions obtained by the yeast two-hybridsystem would reflect direct interactions, as genes encodingappendage proteins are not present in the yeast genome. Full-lengthLRRC45 interacted with Cep83 and SCLT1 constructs (Fig. 5B, redsquares). LRRC45 failed to interact with Cep123 orweakly interactedwith Cep164 (Fig. 5B). LRRC45 C-terminal truncations interactedwith truncated forms of FBF1 carrying coiled-coil domains (Fig. 5B).Interestingly, the leucine-rich repeats of LRRC45 (LRRC45 1-222

aa) strongly associated with the coiled-coil domain of SCLT1(SCLT1 C-terminus, 554-688 aa) but not with the coiled-coildomains of other proteins (Fig. S5), indicating interaction specificityto SCLT1. These results, together with the complete yeast two-hybriddata of appendage components (Fig. S6), led us to propose thatLRRC45 interacts with distal appendage proteins Cep83 and SCLT1,and weakly with Cep164 (Fig. 5C and Fig. S6).

LRRC45 is required forRab8centrosomal localisation but notfor CP110 removalTo determine which step of cilia biogenesis requires LRRC45, wefirst used transmission electron microscopy to visualise the mothercentriole and basal body of non-ciliated cells. Ciliogenesis initiatesat the mother centriole with the fusion of small pre-ciliary vesicles,which fuse to form a large ciliary vesicle that caps the distal end ofthe mother centriole. In control non-ciliated cells, all basal bodiesinspected were capped by a ciliary vesicle (Fig. 6A,B). In a largenumber of non-ciliated LRRC45-depleted cells (63.6%), the basalbodies were associated with a ciliary vesicle, whereas in 36.4% ofbasal bodies, no vesicles were observed (Fig. 6A,B). Thisphenotype indicates that cilia membrane docking and fusioninitiates in the majority of LRRC45-depleted cells upon serum

Fig. 2. Depletion of LRRC45 does not causepremature centrosome separation.(A) Representative images showing 0.5 µm and10 µm intercentrosomal distance in ciliated WTand CEP250−/− RPE1 cells, respectively.ARL13B (green), poly-glutamylated tubulin(pGlu tub), γ-tubulin (red) and DAPI (blue) serveas markers for ciliary membrane, axoneme,basal body and nucleus, respectively. Scale bar:5 µm. (B-D) Quantification of intercentriolardistance in ciliated (blue) and non-ciliated (red)WT (n: 202) and CEP250−/− clone 7 (n=223),and clone 17 (n=229) RPE1 cells, respectively.Cells were grouped according to the distancebetween their centrioles; distance <1 µm,between 1 and 2 µm, and >2 µm. Bar graphsshow the average percentages of cells withineach individual intercentriolar distance group inthree independent experiments. (E)Quantification of intercentriolar distance incycling RPE1 cells treated with siControl (blue)(n=325), and siLRRC45 (red) (n=1383).siLRRC45 represents mean intercentriolardistance measured using siLRRC45 pool,siLRRC45-2 and siLRRC45-3. (F) Quantificationof intercentriolar distance in ciliated (blue) andnon-ciliated (red) serum-starved RPE1 cellstreated with siControl (n=281), and siLRRC45(n=1087). Error bars represent s.d.; *P<0.05;n.s., not significant (Student’s t-test).

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starvation. This conclusion is consistent with the fact that theendosomal protein EHD1, which is required for pre-ciliary vesiclefusion at the mother centriole (Lu et al., 2015), was present in 60%of control cells and in more than 40% of non-ciliated LRRC45-depleted cells (Fig. 6C,D). The association of EHD1 with themother centriole was drastically reduced in the absence of Cep164or Cep123 (Fig. 6C,D), when vesicle docking is severely affected(Schmidt et al., 2012; Sillibourne et al., 2013). Together, these dataimply that LRRC45 plays a role in axoneme extension after ciliarymembrane establishment.We further analysed the key steps in the initiation of primary cilium

formation.We first looked into the CP110-Cep97 complex, which is anegative regulator of ciliogenesis and its removal from the mothercentriole is a pre-requisite for axoneme extension (Spektor et al.,2007). To analyse whether LRRC45 is required for CP110-Cep97removal at the mother centriole, we followed CP110 centrosomallocalisation upon serum starvation (Fig. 6E,F). As expected, CP110did not decorate the basal body of ciliated cells (Fig. 6E, top panel forrepresentative image). In non-ciliated cells, CP110 was absent fromthemajorityof the basal bodies (labelledbyODF2) inboth control andLRRC45-depleted cells (Fig. 6F). The removal of the CP110-Cep97complex was shown to require distal components including Cep164and the kinase TTBK2 (Goetz et al., 2012; Schmidt et al., 2012).Consistently, Cep164 and TTBK2 centrosomal levels were notaffected by LRRC45 depletion (Fig. S6D and S6F). Our data thusindicate that LRRC45 is dispensable for CP110-Cep97 complexregulation at the mother centriole at early stages of ciliogenesis.Transition zone establishment occurs after EHD1 docking to the

mother centriole and removal of CP110 from the mother centriole

(Lu et al., 2015; Yadav et al., 2016). We thus analysed localisationof NPHP1 as a marker of transition zone to investigate whetherLRRC45 influences transition zone formation upon serumstarvation. As expected, NPHP1 was present at the basal body ofcontrol and LRRC45-depleted ciliated cells (Fig. 6G). NPHP1could also be detected at the basal body of non-ciliated cells lackingLRRC45 (Fig. 6G,H) but at lower levels than in the control (Fig. 6I).This implies that LRRC45 may influence transition zoneestablishment. To further investigate this possibility, we analysedthe localisation of Rab8. Upon serum starvation, Rab8 accumulatesaround centrosomes in a manner that requires a functional transitionzone (Bachmann-Gagescu et al., 2015; Kim et al., 2008, 2014). Theenrichment of Rab8 around centrosomes was significantly reducedin non-ciliated LRRC45-depleted cells in comparison to controlcells (Fig. 6J,K). However, the loss of centrosomal Rab8 inLRRC45-depleted cells was not as pronounced as in cells lackingCep164 where no ciliary vesicles, including Rab8a containingvesicles, dock to the mother centriole (Fig. 6K) (Schmidt et al.,2012). These data suggest that LRRC45 is required but not essentialfor Rab8 vesicle docking at the mother centriole upon induction ofcilia biogenesis.

LRRC45 promotes centriolar satellite localisationTo better understand how LRRC45 contributes to ciliogenesis, weasked whether LRRC45 influenced the centrosomal enrichment ofcentriolar satellites, which are large protein assemblies required fortransition zone establishment and cilia formation (Craige et al.,2010; Garcia-Gonzalo et al., 2011; Hori et al., 2014; Hori and Toda,2017; Jin et al., 2010; Kim et al., 2008; Klinger et al., 2013;

Fig. 3. LRRC45 localises to distal but notsub-distal appendages. Representativeimages of LRRC45 co-staining with ODF2(n=133) (A), Cep83 (n=92) (B), Cep164(n=117) (C), SCLT1 (n=118) (D), Cep123(n=78) (E) from top view (top panels) and sideview (bottom panels) using STED microscopy.L.S. (line scan) graphs show the plot profile ofLRRC45 (red) and respective appendageprotein (green) from side view images.Sketches of side views show colocalisation(yellow) of LRRC45 (red) and the indicatedappendage protein (green). Scale bar: 0.5 µm.(F) Table shows mean±s.d. diameter ofLRRC45 ring with respect to Cep164, Cep123and ODF2. LRRC45-Cep164, n=13; LRRC45-Cep123, n=7; LRRC45-ODF2, n=8.

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Kurtulmus et al., 2016). For this, we analysed the centriolar satelliteproteins PCM1 and SSX2IP. In comparison to control cells, thelevels of PCM1 and SSX2IP around centrosomes significantlydecreased in the absence of LRRC45 (Fig. 7A,B and Fig. S7A).Moreover, a similar phenotype of satellite disorganisation wasobserved in cells depleted of Cep83, SCLT1, FBF1 and Cep123(Fig. 7A,B) but not in Cep164-depleted cells (Fig. 7A,B). Theseresults together suggest that distal appendages have a function incentriolar satellite organisation at centrosomes.To test whether loss of satellite organisation could be a

consequence of microtubule loss at centrosomes in response toLRRC45 depletion, we compared the kinetics of microtubulenucleation at centrosomes in control and LRRC45-depleted cells(Fig. S7B). To visualise microtubules, we followed the microtubuleplus-end binding protein EB1 (Mimori-Kiyosue et al., 2000). Coldtreatment depolymerised microtubules and decreased centrosomalEB1 (Fig. S7B). After raising the temperature to 30°C, microtubulesre-formed from centrosomes with similar kinetics in WT andLRRC45-depleted cells (Fig. S7B). Similar results were obtainedfor cells lacking Cep83 (Fig. S7B). This indicates that distalappendage components, including LRRC45, are not required formicrotubule nucleation at centrosomes. Furthermore, the

centrosomal levels of the intraflagellar transport protein IFT88,which is transported to the centrosome in a microtubule-dependentmanner (Kozminski et al., 1993; Pazour et al., 1998), were notdecreased by depletion of LRRC45 (Fig. S6G). This indicates thatLRRC45 does not impair microtubule-dependent transport tocentrosomes in general.

DISCUSSIONAppendage proteins at the mother centriole play a critical roleduring the initial steps of cilia formation. Here, we have identifiedLRRC45 as a component that associates with distal appendages thatcontributes to the early phases of ciliogenesis (Fig. 7C). Given thatLRRC45 localises to the basal body in diverse cell lines, we proposethat the function of LRRC45 is conserved from cells that form asingle primary cilium, such as stem and differentiated cells, to cellswith multiple motile cilia.

LRRC45 is recruited to the proximal ends of mother and daughtercentrioles by the linker protein C-Nap1. Super-resolution andquantitative fluorescence microscopy indicate that LRRC45 bindsto the distal appendage of the mother centriole in a manner thatrequires Cep83 and SCLT1. Proximal and distal pools of LRRC45co-exist at the mother centrioles and association of LRRC45 with

Fig. 4. LRRC45 bridges Cep83-SCLT1 and FBF1 in the hierarchy of distal appendage proteins. (A) LRRC45 (green) localisation in control and Cep83-depletedCEP250−/−RPE1 cells. γ-tubulin (red) and DAPI (blue) serve asmarkers for centrosomes and nuclei, respectively. (B) Bar graph shows the centrosomalLRRC45 mean fluorescence intensity in three independent experiments upon control and Cep83 depletion in CEP250−/− RPE1 cells (n=299 for siControl andn=419 for siCep83). LRRC45 mean fluorescence in siControl-treated cells is normalised to 100. (C) LRRC45 (red) and SCLT1 (green) localisation in control andSCLT1-depleted CEP250−/− RPE1 cells. γ-tubulin (grey) and DAPI (blue) served as markers for centrosomes and nuclei, respectively. (D) Bar graph shows thecentrosomal LRRC45 mean fluorescence intensity in three independent experiments upon control and SCLT1 depletion in CEP250−/− RPE1 cells (siControl,n=321; siSCLT1, n=546). LRRC45 mean fluorescence in siControl-treated cells is normalised to 100. (E) LRRC45 (red) and FBF1 (green) localisation in controland LRRC45-depleted RPE1 cells. γ-tubulin (grey) and DAPI (blue) serve as markers for centrosomes and nuclei, respectively. (F) Bar graph shows thecentrosomal FBF1 mean fluorescence intensity in three independent experiments upon control (n=436) and LRRC45 (n=1179) depletion. siLRRC45 representsthe mean values of FBF1 fluorescence intensities measured using siLRRC45 pool, siLRRC45-2 and siLRRC45-3. FBF1 mean fluorescence in siControl-treatedcells is normalised to 100. (G) Model for position of LRRC45 in the hierarchy of distal appendage proteins (Tanos et al., 2013). Error bars represent s.d.;***P<0.001 (Student’s t-test).

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distal appendages does not depend upon C-Nap1. This implies thatC-Nap1 and Cep83-SCLT1 do not compete for LRRC45 binding.Distal appendage proteins were recently proposed to form twospatially distinct domains, named distal appendage blades andmatrix. According to this model, Cep83, SCLT1, Cep164 andCep123 form defined conical-shaped blades whereas FBF1 makesup the matrix by filling the space between the blades (Yang et al.,2018). The reduced level of FBF1 at the mother centriole observedin the absence of LRRC45 indicates that LRRC45 is a keydeterminant of FBF1 centriolar localisation. It is therefore temptingto speculate that LRRC45 might be part of the appendage bladecomplex that is recruited via Cep83 and SCLT1 to help maintainFBF1 at this location.Recently, the proximity of the daughter to the mother centriole

was proposed to be important for ciliogenesis (Loukil et al., 2017).Our data indicate that centriole separation cannot be the underlyingmechanism explaining the lack of cilia formation in LRRC45-depleted cells. First, the depletion of LRRC45 did not cause asignificant separation of mother and daughter centrioles in RPE1serum-starved cells, even though these cells did not form cilia.Second, in CEP250 KO cells, which lose the LRRC45 proximalpool (Fig. 1) (He et al., 2013), LRRC45 remained at the distal endsof the mother centriole. In those cells, cilia were formed in a

LRRC45-dependent manner even when centrioles were more than8 µm apart. Therefore, our data do not show any correlation betweencilia loss and distance between centrioles. We thus propose thatLRRC45 plays a key function in ciliogenesis at distal appendages.

Ciliogenesis is a tightly regulated process that occurs at theG1/G0 phase of the cell cycle and involves the conversion of themother centriole into the basal body. One critical step in this initialphase is the docking of small vesicles at the distal appendages of themother centriole, which then fuses to generate a large vesicle thatcaps the distal end of this centriole (Graser et al., 2007; Joo et al.,2013; Kurtulmus et al., 2016; Schmidt et al., 2012; Sillibourne et al.,2013; Sorokin, 1962; Tanos et al., 2013; Ye et al., 2014). Vesiclefusion is promoted by components of recycling endosomes,including the transmembrane tethering protein EHD1 (Lu et al.,2015). Subsequent steps involve membrane elongation, removal ofinhibitory components from the mother centriole and targeteddelivery of proteins to the basal body (via IFT- and centriolarsatellite-dependent transport) to allow axoneme extension (Fig. 7C)(Sánchez and Dynlacht, 2016). Interestingly, defects at early stagesof membrane establishment block CP110-Cep97 removal andaxoneme elongation, implying that these are highly coordinatedprocesses. The common concept is that distal appendages are keycomponents for this coordination, yet how this is achieved on a

Fig. 5. LRRC45 interacts with Cep83, SCLT1 and Cep164in the yeast two-hybrid system. (A) Schematicrepresentation of existing domains in appendage proteins.Numbers represent amino acid positions. The dashed linesindicate where truncated constructs were created. LRR,leucine-rich repeat domain. (B) Yeast two-hybrid assay formapping the interactions between LexA and Gal4 fusionproteins, as indicated. Development of blue colour indicatesinteraction. Full set of interactions is shown in Fig. S5.Interacting full-length proteins are marked with a red square.(C) Model of the interaction map of distal appendagesaccording to yeast two-hybrid assay. Dashed lines of thesame colour encircle interacting full-length proteins.

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Fig. 6. LRRC45 is required for recruitment of Rab8a-positive vesicles to the mother centrosome. (A) Representative electron micrographs of 70 nm serialsections showing ciliary vesicle docked mother centrioles in control and LRRC45-depleted RPE1 cells (using siLRRC45-2 and siLRRC45-3 in combination). DA,distal appendage; SDA, subdistal appendage; CV, ciliary vesicle. Scale bar: 200 nm. (B) Table showing percentage of cells with centrosomes having no anddocked ciliary vesicles observed in A. n, total number of centrosomes analysed for respective conditions. (C) RPE1 mNeongreen-EHD1 cells with docked orundocked EHD1 (green) foci. Images were taken after 16 h of serum starvation. ARL13B (grey), γ-tubulin (red) and DAPI (blue) serve as markers for ciliarymembrane, centrosomes and nuclei, respectively. Scale bars: 5 µm and 1 µm (insets). (D) Bar graph shows the percentage of non-ciliated cells containing EHD1foci at their centrosomes upon control (n=141), LRRC45 (n=560), Cep123 (n=349) and Cep164 (n=386) depletion and serum starvation for 16 h, in threeindependent experiments. siLRRC45 represents the mean values for EHD1 docking observed using siLRRC45 pool, siLRRC45-2 and siLRRC45-3. (E)Localisation of CP110 (green) at the mother centriole in ciliated and non-ciliated RPE1 after 16 h of serum starvation. ODF2 (red) and ARL13B (grey) serve asmarkers for mother centriole and cilia, respectively. Scale bar: 2 µm. (F) Bar graph shows the percentage of mother centrioles with CP110 removed in non-ciliatedRPE1 cells in control (blue, n=94) and after LRRC45 depletion (red, n=591) and serum starvation for 16 h, in three independent experiments. siLRRC45represents the mean values for siLRRC45 pool, siLRRC45-2 and siLRRC45-3. (G) NPHP1 (red) localisation in ciliated and non-ciliated RPE1 cells upon controland LRRC45 depletion. ARL13B (green), γ-tubulin (green) and DAPI (blue) serve as markers for ciliary membrane, centrosomes and nuclei, respectively. Scalebars: 5 and 1 µm (insets). (H) Bar graph shows the percentage of non-ciliated cells showing NPHP1 localisation at their centrosomes upon control and LRRC45depletion. siLRRC45 represents mean values observed using siLRRC45 pool, siLRRC45-2, and siLRRC45-3 individually. (I) Bar graph shows the centrosomalNPHP1 mean fluorescence intensity (analysed in H) measured in non-ciliated RPE1 cells upon control (n=140) and LRRC45 (n=839) depletion after 48 h serumstarvation, in three independent experiments. NPHP1mean fluorescence in siControl-treated cells is normalised to 100. (J) RPE1 EGFP-Rab8a cells with dockedor undocked Rab8a (green) foci at the centrosomes. Images were taken after 16 h of serum starvation. ARL13B (grey), γ-tubulin (red) and DAPI (blue) serve asmarkers for ciliary membrane, centrosomes and nuclei, respectively. Scale bar: 2 µm. (K) Bar graph shows the percentage of cells lacking an elongated cilia thatcontain docked Rab8a at their mother centrioles upon control (n=301), LRRC45 (n=869) and Cep164 (n=281) depletion and serum starvation for 16 h, in fourindependent experiments. siLRRC45 represents mean values for Rab8a docked centrosomes using siLRRC45 pool, siLRRC45-2 and siLRRC45-3. Error barsrepresent s.d.; **P<0.01, ***P<0.001; n.s., not significant (Student’s t-test).

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molecular level remains unclear. Our analyses show that, unlikeCep164 or Cep123, LRRC45 is not a major determinant for EHD1centrosome accumulation or initial steps of ciliary vesicle docking. Inthe absence of LRRC45, the ciliary vesicle capped the majority ofbasal bodies; however, axonemeand ciliarymembrane extensionwereblocked. The lack of axoneme extension in the absence of LRRC45was not due to defects in CP110-Cep97 removal, as the majority ofnon-ciliated LRRC45-depleted cells had CP110-uncapped mothercentrioles. This is in agreement with the fact that LRRC45 did notaffect the ability of Cep164 to recruit the kinase TTBK2 to promoteCP110 centrosomal removal (Goetz et al., 2012). LRRC45 also didnot influence the centrosome levels of IFT88, which is required topromote axoneme extension (Sánchez and Dynlacht, 2016).Our data indicate that the inability of LRRC45-depleted cells to

ciliate is related to defective Rab8-dependent ciliary membraneextension, transition zone formation and centriolar satelliteorganisation at centrosomes. In the absence of LRRC45, thelevels of the satellite proteins PCM1 and SSX2IP aroundcentrosomes were significantly reduced. Similar results wereobserved after depletion of Cep83, SCLT1, Cep123 and FBF1 but

not Cep164. Centriolar satellites have been implicated in transitionzone establishment and targeting of Rab8-vesicles to the mothercentriole upon induction of ciliogenesis (Hori et al., 2014; Kimet al., 2008; Klinger et al., 2013; Kurtulmus et al., 2016; Lopes et al.,2011; Nachury et al., 2007; Wang et al., 2016). It is thus tempting tospeculate that a yet-to-be-identified component required foraxoneme extension is targeted to the forming basal body byRab8-positive vesicles. Alternatively, centriolar satellites might berequired for centriolar microtubule remodelling to promoteaxoneme extension after removal of CP110-Cep97. Futureresearch should thus concentrate on understanding how thefunction of centriolar satellites and Rab8-vesicles are coordinatedby distal appendage components at early stages of ciliogenesis.

We propose that appendages can be sub-divided into threecategories or modules based on their function in (I) promotingdocking of small ciliary vesicles, (II) recruiting TTBK2 andremoving inhibitory CP110-Cep97 complexes and (III) promotingRab8-positive vesicle docking to initiate membrane extension andaxoneme elongation in addition to satellite organisation (Fig. 7C).Both the Cep83-Cep123 and Cep83-SCLT1-Cep164 branch of

Fig. 7. LRRC45 is required for satelliteorganisation. (A) Localisation of PCM1(green) in RPE1 cells upon control,Cep83, SCLT1, FBF1, LRRC45, Cep164and Cep123 depletion. γ-tubulin (red) andDAPI (blue) serve as markers forcentrosomes and nuclei, respectively.(B) Bar graph shows PCM1 meanfluorescence intensities around thecentrosomes in control (n=244) and inCep83 (n=371), SCLT1 (n=530), FBF1(n=332), LRRC45 (n=583), Cep164(n=170) and Cep123 (n=213) depletedRPE1 cells, in three independentexperiments. siLRRC45 represents themean values observed using siLRRC45-2 and siLRRC45-3 individually. Scalebars: 10 µm and 2 µm (insets). Error barsrepresent standard deviation. **P<0.01,***P<0.001; n.s., not significant(Student’s t-test). (C) Model for the role ofLRRC45 in ciliogenesis. LRRC45 isrecruited to the centrosomes via Cep83-SCLT1, and is required for FBF1localisation at the distal appendages,recruitment of transition zone, docking ofRab8a-positive vesicles and organisationof centriolar satellites.

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distal appendages, are required for the early steps of small vesicledocking (Graser et al., 2007; Joo et al., 2013; Schmidt et al., 2012;Sillibourne et al., 2013; Tanos et al., 2013). Cep83-Cep123 has anadditional function in centriolar satellite organisation (Sillibourneet al., 2013), which is not shared by the Cep83-SCLT1-Cep164complex (Cajanek and Nigg, 2014; Graser et al., 2007; Oda et al.,2014; Schmidt et al., 2012). Without both modules, subsequentsteps related to TTBK2 recruitment, CP110 removal, Rab8-vesicledocking and axoneme extension are blocked. In contrast, the Cep83-SCLT1-LRRC45-FBF1 module works downstream in this cascadeof events to promote Rab8 vesicle docking, transition zoneestablishment as well as satellite organisation (Fig. 7C) (Graseret al., 2007; Joo et al., 2013; Schmidt et al., 2012; Sillibourne et al.,2013; Tanos et al., 2013; Wei et al., 2013; Ye et al., 2014). Cep164might also assist in this process, given that Cep164 interacts withcomponents of the Rab8 machinery (Schmidt et al., 2012; Burkeet al., 2014). Importantly, the LRRC45 siRNA phenotype is distinctfrom those seen upon depletion of other distal appendage proteins,suggesting that centriole appendages are not simply a scaffold forvesicle recruitment but provide a multi-task platform of cooperating,rearranging proteins with defined functions during ciliogenesis.

MATERIALS AND METHODSPlasmids and reagentsHsLRRC45 (BC014109), HsCep83 (NM_016122), HsSCLT1 (BC128051)and OFD1 (BC096344) were amplified from an RPE1 cDNA library;MmLRRC45 (BC023196) was amplified from an NIH 3T3 cDNA library.Briefly, total mRNA from RPE1 or NIH3T3 cells was isolated usingRNeasy Mini Kit (Qiagen) according to the manufacturer’s protocol, andused for cDNA synthesis using RevetAid First Strand cDNA Synthesis Kit(Thermo Fisher Scientific). HsFBF1 (BC023549) and HsEHD1(BC104825) cDNAs were purchased from Dharmacon (clones 4109753and 8143828, respectively). HsCep123 cDNA was a gift from MichelBornens, Curie Institute, Paris, France (Sillibourne et al., 2013). Coiled-coildomains of the proteins were determined using the COILS predictionprogramme (Lupas et al., 1991), and truncations were cloned into yeast two-hybrid vectors pMM5, pMM6 and frame-modified pMM5 and pMM6(Schramm et al., 2001). For rescue experiments, MmLrrc45 (from mousecDNA), IRES2 (from pQCXIP-GFP) and GFP (from pEGFP-C1) werecloned into pRetrox-TRE3G (Takara Bio) using NEB HiFi assembly (NewEngland Biolabs). HsEHD1 was cloned into pMSCV-Zeo-C1-mNeonGreen [pMSCV-Zeocin, Addgene plasmid #75088 deposited byDavidMu (Kendall et al., 2007)]. More detailed information can be found inTable S1.

Cell culture and transfectionh-TERT-immortalised retinal pigment epithelial (RPE1, ATCC, CRL-4000,USA) cells were grown in DMEM/F12 (Sigma Aldrich) supplemented with10% fetal bovine serum (FBS, Biochrom), 2 mM L-glutamine (ThermoFischer Scientific) and 0.348% sodium bicarbonate (Sigma Aldrich).Medium of RPE1 cells stably expressing GFP-Rab8a was supplementedwith G418 (500 µg/ml, Thermo Fischer Scientific) (Schmidt et al., 2012).NIH 3T3 cells (ATCC, CRL-1658) were grown in DMEM high glucose(Sigma Aldrich) supplemented with 10% newborn calf serum (NCS, PAN-Biotech). HEK293T (ATCC CRL-3216) and GP2-293 cells (Takara Bio)were cultured in DMEM high glucose supplemented with 10% FBS. Adultneuronal stem cells were isolated from sub-ventricular zone of 8-week-oldC57BL/6 mouse and cultured in DMEM/F12 supplemented with 0.236%sodium bicarbonate, 6% Glucose (Sigma Aldrich), 50 mM HEPES (SigmaAldrich), 3.14 µg/ml progesterone (Sigma Aldrich), 4.8 µg/ml putrescinedihydrochloride (Sigma Aldrich), 0.37 U/ml heparin (Sigma Aldrich), 2%B-27 supplement (Thermo Fischer Scientific), 0.1% insulin, transferrin,sodium selenite supplement (ITSS, Sigma Aldrich), 10 ng/ml fibroblastgrowth factor-basic (FGF-2, Sigma Aldrich), 20 ng/ml epidermal growthfactor (EGF, Sigma Aldrich). All cell lines were grown at 37°C with 5%CO2. Transient transfection in HEK293T and GP2-293 cells was performed

using polyethyleneimine (PEI 25000, Polysciences). RPE1 cells weretransiently transfected with plasmid DNA by either electroporation using theNeon® Transfection System or TurboFect (Thermo Fisher Scientific)according to the manufacturer’s protocol. Stable cell lines expressingMmLrrc45 and HsEHD1 were generated using retroviral-mediatedtransduction. GP2-293 cells were transiently transfected with pRetrox-TRE3G-MmLrrc45-IRES2-GFP or pMSCV-Zeo-C1-mNeonGreen-HsEHD1. 48 h after transfection, supernatant was used to transduce RPE1cells. Expression of TET-ON inducible constructs was induced by theaddition of doxycycline (Sigma Aldrich) at a concentration of 1-10 ng/ml.RPE1 CEP250 knockout cell lines were described previously (Panic et al.,2015). To induce cilia formation, 1.2×105-1.5×105 RPE1 or NIH 3T3 cellswere seeded on coverslips in 6-well plates and incubated in serum-freemedium for 16-48 h. Cell number was determined using the Luna automatedcell counter (Logos Biosystems). At least 100 cells were counted for thequantification of ciliated cells for each experimental condition. All celllines used in this study were authenticated by Multiplex Cell lineAuthentication (MCA) and were tested for contamination by Multiplexcell Contamination Testing (McCT) service provided by Multiplexion(Heidelberg, Germany).

AntibodiesAntibodies against human LRRC45 (rabbit, 1:800 and guinea pig, 1:400)were produced against C-terminal recombinant protein (240-670 aa) inrabbits and guinea pigs. In brief, 6×His-tagged antigen was purified fromBL21 and used to immunise animals. For purification of the antibodies,GST-LRRC45-C (240-670 aa) was used. Antibodies against human Cep123(1:400) were produced against the N-terminus of Cep123 (1-230 aa)(Sillibourne et al., 2011) in guinea pigs. Recombinant protein GST-Cep123-N (1-230 aa) was used for purification of the antibodies. Specificity of theantibodies was tested by immunofluorescence depletion of LRRC45 orCep123 using siRNAs in RPE1 cells.

Primary antibodies used for indirect immunofluorescence were rabbit andguinea pig polyclonal anti-Cep164 (1:500 and 1:2000, respectively;Schmidt et al., 2012); rabbit and guinea pig polyclonal anti-ODF2 (1:100and 1:500, respectively; Kuhns et al., 2013); guinea pig polyclonalanti-IFT88 (1:250; Gazea et al., 2016). Mouse monoclonal anti-polyglutamylated tubulin clone GT335 (1:2000) was a gift from CarstenJanke (Institute Curie, Paris, France) (Wolff et al., 1992). Mousemonoclonal anti-Chibby (1:100) was a gift from Ryoko Kuriyama(Department of Genetics, Cell Biology and Development at theUniversity of Minnesota, USA) (Burke et al., 2014). Rabbit polyclonalantibodies anti-PCM1 (1:2000) and anti-SSX2IP (1:250) were a gift fromOliver Gruss (Institute of Genetics, University of Bonn, Germany) (Bärenzet al., 2013).

Antibodies from commercial sources used in this study were as follows:rabbit polyclonal and mouse monoclonal anti-ARL13B (1:500, Proteintech,#17711-1-AP and 1:50, UC Davis/NIH NeuroMab Facility, clone N295B/66, #73-287, respectively); mouse monoclonal anti-γ-tubulin clone GTU-88(1:500, Sigma Aldrich, #T6557); rabbit polyclonal anti-TTBK2 (1:1000,Sigma Aldrich, #HPA018113); rabbit polyclonal anti-Cep83 (CCDC41)(1:400, Sigma Aldrich, #HPA038161); rabbit polyclonal anti-SCLT1(1:250, Sigma Aldrich, #HPA036561); rabbit polyclonal anti-FBF1(1:500, Sigma Aldrich, #HPA023677); rabbit polyclonal anti-MAPRE1(EB1) (1:300, Abcam, #ab53358); rabbit polyclonal anti-CP110 (1:300,Bethyl Laboratories, #A301-343A).

RNA interferenceTransfections of siRNA were performed using Lipofectamine RNAiMAXtransfection reagent (Thermo Fischer Scientific). siRNA transfections wereperformed briefly as following, 1.2×105 cells were reverse-transfected inone well of a 6-well plate. For double depletion, 48 h after the initialtransfection, cell were dissociated, counted and 1.5×105 cells were seeded in6-well plates and reverse transfected for the second time. For LRRC45 andCep83 depletions, double depletion method was used. In experiments whereLRRC45 depletion was compared to depletion of other proteins, doubledepletion method was used. Cycling cells were analysed 48 h hours after thesecond knockdown. For cilia analysis, 16 h after second transfection, cells

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were serum starved for 48 h, if not stated otherwise. Phenotypes observed byLRRC45 depletion using individual LRRC45 siRNAs were averaged andshown as single value. Detailed information about the siRNAs, along withfinal molar concentrations used in the study, can be found in Table S2.

Rescue experimentsRPE1 TRE3G-MmLrrc45-IRES2-GFP cells were incubated in mediumcontaining 0, 1, 5 or 10 ng/ml doxycycline for 6 h prior to LRRC45 doubledepletion. Cells were then maintained in doxycycline-containing medium.

Indirect immunofluorescence and microscopyCells were grown on coverslips (No. 1.5, Thermo Fischer Scientific) andfixed in ice-cold methanol at−20°C for 10 min. Cells expressing fluorescentprotein fusions were fixed with 3% PFA for 3 min prior to methanolfixation. Coverslips were coated with 0.1 mg/ml collagen A (Biochrom) forculturing NIH 3T3 cells. For adult NSCs, coverslips were coated with0.1 mg/ml poly-L-lysine (Sigma Aldrich) and 0.05 mg/ml laminin (SigmaAldrich) sequentially. Cells were blocked with blocking solution containing3% IgG-free BSA (Jackson ImmunoResearch), 0.1% Triton X-100 (SigmaAldrich) in PBS for 30 min and incubated with primary antibodies at 37°Cfor 1 h. The cells were then incubated with Alexa Fluor 488-, 546-, 594- or647-conjugated secondary antibodies (Thermo Fisher Scientific) togetherwith DAPI (4′,6-diamidino-2-phenylindole) for 45 min at roomtemperature. All antibodies were diluted in blocking solution. Coverslipswere mounted with Mowiol (EMD Millipore).

Images were acquired as Z stacks using either Zeiss Axiophot equippedwith 63× NA 1.4 Plan-Fluor oil immersion objective, and Cascade:1KEMCCD camera using Meta.Morph software, or using Zeiss Axio ObserverZ1 equipped with 63× NA 1.4 Plan-Apochromat oil immersion objective,and AxioCam MRm CCD camera using ZEN software.

3D-SIM images were acquired as Z-stacks using a Nikon Ti invertedmicroscope equipped with 488 nm, 561 nm and 647 nm laser lines, a NikonApo TRIF 100× NA 1.49 oil immersion objective and an Andor iXon3DU-897E single photon detection EMCCD camera. After acquisition,images were reconstructed using NIS Elements program.

STED images were acquired on with STEDYCON STED microscopeplatform mounted on Zeiss Axio Imager Z2 with a 100× NA 1.46 oilimmersion objective. All images were taken at room temperature. Proteinsfused to fluorescent proteins were visualised by the fluorescence signal.Figures were assembled in Adobe Photoshop and Illustrator CS3 (Adobe).

Preparation and imaging of brain tissueEight-week-old C57BL/6J mice (Jackson Laboratory) were euthanised andbrains were extracted and fresh-frozen in Tissue-Tek OCT compound(Sakura FineTek). Coronal sections of 12 µm were cut on a Leicacryomicrotome. Sections were permeabilised with PBS containing 0.1%Triton X-100 prior to fixation with ice-cold methanol at −20°C for 30 minand immunostaining was performed with overnight primary and secondaryantibody incubations at 4°C. Sections were imaged on a Nikon A1Rconfocal microscope equipped with Nikon N Apo 60× NA 1.4 λs oilimmersion objective, using NIS Elements software. Mice were obtainedfrom the Deutsches Krebsforschungszentrum (DKFZ) Centre for PreclinicalResearch facility, housed under standard conditions of 12 h:12 h light:darkand fed ad libitum. All procedures were in accordance with the DKFZguidelines and approved by the Regierungspräsidium Karlsruhe.

Transmission electron microscopyRPE1 cells were grown on coverslips, rinsed with PBS and fixed with amixture of 2.5% glutaraldehyde, 1.6% paraformaldehyde for 30 min at roomtemperature. The fixative was washed out with 50 mM cacodylate buffer.After post-fixation in 2% OsO4 for 1 h at 4°C, cells were incubated in 0.5%aqueous uranyl acetate overnight, rinsed in water and then dehydrated atroom temperature using ascending ethanol concentrations. The coverslipswere subsequently set on spurr (Serva)-filled capsules and polymerisedovernight at 60°C for 48 h. Serial ultrathin sections (70 nm) were poststained with uranyl acetate and lead citrate and examined at a JEM-1400electronmicroscope (JEOL, Tokyo), operating at 80 kV and equipped with a

4K TemCam F416 (Tietz Video and Image Processing Systems GmBH,Gautig). Image brightness and contrast were adjusted in ImageJ.

Microtubule re-growthCells grown on coverslips were incubated on ice for 2 h in order todepolymerise microtubules. Cells were fixed in ice-cold methanol afterincubation at 30°C for 0, 5 and 20 s, respectively.

Yeast two-hybrid assayFull-length or partial ORF of the indicated genes were cloned into pMM5(LexA) and pMM6 (Gal4), and the assay was performed as described(Geissler et al., 1996). Yeast growth conditions were as described (Sherman,1991). Briefly, plasmids encoding LexA and Gal4 fusion proteins weretransformed into yeast strains that have opposite mating types and selectedaccording to their auxotrophy markers. A pool of colonies (more than 30colonies) from the transformation plates were picked and streaked asindividual lines on selection plates. Transformants of LexA fusion proteinswere then mated with the Gal4 fusion proteins on non-selective plates for2 days, and the diploid strains were selected on double selection plates. Twoto three days after selection, the plates were covered with Overlay Solutioncontaining 0.25 M sodium phosphate buffer pH 7.0, 0.1% SDS, 10 mMKCl, 1 mM MgCl2, 0.4% low melting agarose and 0.04% X-Gal.Development of blue colour was observed and recorded. The assay withthe complete set of genes was repeated twice.

Image processing and analysisQuantification of fluorescence intensity was performed using maximumprojection of images using Fiji and CellProfiler (Carpenter et al., 2006;Schindelin et al., 2012; Schneider et al., 2012). Briefly, images werecorrected for background signal by applying a top hat filter, and thencentrosomes were segmented using γ-tubulin signal. Identified centrosomeareas were expanded by 2 pixels and 10 pixels for intensity measurement ofcentrosomal proteins and centriolar satellites, respectively. Intensitymeasurement from replicate experiments were normalised and combined.Intercentriolar distances were manually measured using Fiji. Statisticalanalyses of fluorescence intensity measurements and ciliation assays wereperformed using Wilcoxon and two-tailed Student’s t-tests; significanceprobability values are: *P<0.05, **P<0.01, ***P<0.001. Statistical testswere performed in Excel (Microsoft) and KaleidaGraph (SynergySoftware).

FACS analysisCell cycle profile of control and LRRC45-depleted serum-starved RPE1cells were determined by analysing total DNA content using propidiumiodide staining. Briefly, cells were fixed with ice-cold ethanol (70%), andincubated with staining solution (50 µg/ml propidium iodide, 0.08% TritonX-100, 0.2 mg/ml RNase A, in PBS/1 mM EDTA). Cells were subjected toanalysis on a BD FACS Canto.

AcknowledgementsWewould like to thank to Marko Panic, Carsten Janke, Michel Bornens, Andrew Fry,Ryoko Kuriyama andOliver Gruss for sharing reagents; Astrid Hofmann for excellenttechnical support; Rafael Duen as-Sanchez for help with antibody production; BeratiCerikan for working on the initial stage of the project and cloning SCLT1 and Cep83;Monika Langlotz from the ZMBH FACS facility for help with the cell cycle analysis,Damir Krunic and Felix Bestvater from themicroscopy facility of the DKFZ, andUlrikeEngel from the Heidelberg-University Nikon Imaging Center for providing access tomicroscopes, help with imaging and image processing. We are grateful to AbberiorInstruments GmbH for allowing us access to the STEDYCON STED microscopeplatform.

Competing interestsThe authors declare no competing or financial interests.

Author contributionsConceptualization: B.K., G.P.; Methodology: B.K., G.K., G.P.; Software: B.K.;Validation: B.K., G.P.; Formal analysis: B.K., G.P.; Investigation: B.K., C.Y., J.S.,A.N.; Resources: B.K., S.H., G.K., A.M.-V., G.P.; Data curation: B.K.; Writing -original draft: B.K., G.P.; Writing - review & editing: B.K., C.Y., J.S., A.N., S.H., G.K.,

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A.M.-V., G.P.; Visualization: B.K.; Supervision: G.P.; Project administration: G.P.;Funding acquisition: G.P.

FundingThis project was funded by the collaborative research grant from the DeutscheForschungsgemeinschaft (DFG) (SFB873) granted to G.P. (Project A14). Corefunding for microscopy was provided by the SFB873, Project Z03. B.K. is a memberof the Heidelberg Biosciences International Graduate School (HBIGS) and fundedby the SFB873 grant. C.Y. is a member of the Helmholtz International GraduateSchool (HIGS). G.P. holds a Heisenberg Professorship from the DFG (PE1883/3).

Supplementary informationSupplementary information available online athttp://jcs.biologists.org/lookup/doi/10.1242/jcs.223594.supplemental

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