Ahi1 Promotes Arl13b Ciliary Recruitment, Regulates Arl13b Stability and Is Required for Normal Cell Migration Jesúsmuñoz-Estrada1 and Russell J

RESEARCH ARTICLE Ahi1 promotes Arl13b ciliary recruitment, regulates Arl13b stability and is required for normal cell migration JesúsMuñoz-Estrada1 and Russell J. Ferland1,2,*

ABSTRACT (TZ), and participates in the formation of primary cilia in epithelial Mutations in the Abelson-helper integration site 1 (AHI1) gene are cells (Hsiao et al., 2009). Recently, JBTS has been proposed to associated with neurological/neuropsychiatric disorders, and cause result from disruption of the ciliary TZ architecture, leading to the neurodevelopmental ciliopathy Joubert syndrome (JBTS). Here, defective ciliary signaling (Shi et al., 2017). we show that deletion of the transition zone (TZ) protein Ahi1 in The primary cilium, a slender microtubule-based extension mouse embryonic fibroblasts (MEFs) has a small effect on cilia (axoneme) of the cell membrane, is critical for embryonic formation. However, Ahi1 loss in these cells results in: (1) reduced development and tissue homeostasis (Goetz and Anderson, 2010). localization of the JBTS-associated protein Arl13b to the ciliary In non-dividing cells that form cilia, migration and docking of the membrane, (2) decreased sonic hedgehog signaling, (3) and an basal body (a modified mother centriole) to the apical membrane, abnormally elongated ciliary axoneme accompanied by an increase intraflagellar transport (IFT) and microtubule dynamics are required in ciliary IFT88 concentrations. While no changes in Arl13b levels are for assembly and elongation of the axoneme (Rosenbaum and detected in crude cell membrane extracts, loss of Ahi1 significantly Witman, 2002; Sorokin, 1962; Stephens, 1997). IFT is an reduced the level of non-membrane-associated Arl13b and its evolutionary conserved transportation system powered by IFT stability via the proteasome pathway. Exogenous expression of particles and molecular motors moving structural and functional Ahi1–GFP in Ahi1−/− MEFs restored ciliary length, increased ciliary components into and out of the cilium (Kozminski et al., 1993; recruitment of Arl13b and augmented Arl13b stability. Finally, Ahi1−/− Rosenbaum and Witman, 2002). Between the basal body and cilium MEFs displayed defects in cell motility and Pdgfr-α-dependent lies the TZ, a subdomain that selectively controls the entrance and migration. Overall, our findings support molecular mechanisms exit of ciliary components (Reiter et al., 2012). The TZ is thought to underlying JBTS etiology that involve: (1) disruptions at the TZ restrict lateral diffusion of ciliary membrane components to the resulting in defects of membrane- and non-membrane-associated remaining plasma membrane (Chih et al., 2011; Hu et al., 2010; proteins to localize to primary cilia, and (2) defective cell migration. Williams et al., 2011), thereby maintaining a distinct protein composition between these two cellular compartments. This article has an associated First Person interview with the first ADP-ribosylation factor-like protein-13b (Arl13b) is a ciliary author of the paper. membrane-associated GTPase, mutations in which cause defects in ciliary architecture, ciliogenesis and sonic hedgehog (Shh) signaling KEY WORDS: Ahi1, Arl13b, Cilia, Migration, Shh, Stability (Caspary et al., 2007; Larkins et al., 2011; Mariani et al., 2016). The canonical Shh pathway acts through the secreted glycoprotein Shh, INTRODUCTION and controls embryonic development. When Shh signaling is not Mutations in the Abelson-helper integration site 1 (AHI1) gene are active, the membrane receptor Patched1 (Ptch1) localizes to cilia, one of the more common genetic causes of the neurodevelopmental inhibits the activation of the G protein-coupled receptor Smoothened disorder Joubert syndrome (JBTS). JBTS is characterized by (Smo) and regulates the activity of Gli transcription factors. Once Shh midbrain/hindbrain malformations and a broad spectrum of clinical binds Ptch1, it is inactivated via cellular internalization. Smo is then features, involving other organ systems (Brancati et al., 2010; constitutively trafficked to the primary cilium, leading to upregulation Dixon-Salazar et al., 2004; Ferland et al., 2004; Parisi et al., 2007). of Gli1 and Ptch1 mRNAs (Bai et al., 2002; Corbit et al., 2005; Denef The majority of identified AHI1 pathogenic variants in JBTS result et al., 2000; Rohatgi et al., 2007). in truncated/non-functional protein products (Valente et al., 2006). In addition to ciliary Arl13b regulating transcriptional Shh AHI1 variants have also been associated with neuropsychiatric signaling, Arl13b has also been implicated in interneuron migration disorders, including schizophrenia and autism (Alvarez Retuerto during brain development and in MEF migration (Higginbotham et al., 2008; Ingason et al., 2010). The AHI1 gene encodes for the et al., 2012; Mariani et al., 2016). Missense mutations in ARL13B Ahi1 protein, which contains WD40 repeats and an SH3 domain that result in altered Arl13b function have been identified in (Jiang et al., 2002). Subcellularly, Ahi1 preferentially localizes to individuals with JBTS (Cantagrel et al., 2008; Rafiullah et al., the distal end of the mother centriole, including the transition zone 2017). Individuals with JBTS can also present with neuronal migration disorders, including periventricular, interpeduncular, cortical, and other hindbrain heterotopias (Doherty, 2009; Harting 1Department of Neuroscience and Experimental Therapeutics, Albany Medical et al., 2011; Poretti et al., 2011; Tuz et al., 2014). Finally, mutations College, Albany, NY 12208, USA. 2Department of Neurology, Albany Medical College, Albany, NY 12208, USA. in AHI1 in JBTS have been linked to polymicrogyria, a late neurodevelopmental stage migration disorder (Dixon-Salazar et al., *Author for correspondence ([email protected]; [email protected]) 2004; Gleeson et al., 2004). R.J.F., 0000-0002-8044-2479 Despite the known participation of Ahi1 in primary cilia biogenesis, its participation at the ciliary TZ and in mediating cell

Received 4 February 2019; Accepted 24 July 2019 migration remains elusive. The present study sought to further Journal of Cell Science

1 RESEARCH ARTICLE Journal of Cell Science (2019) 132, jcs230680. doi:10.1242/jcs.230680 investigate the involvement of Ahi1 in cilia function using Ahi1−/− Ahi1 facilitates ciliary recruitment of Arl13b and Htr6, and its MEFs. Consistent with a role for Ahi1 in TZ function, Ahi1 deletion upregulates ciliary length depletion in MEFs disrupts ciliary trafficking of Arl13b and Disruption of TZ components cause defects in ciliary protein reduces Shh signaling. Ahi1−/− cells also display abnormally composition and differences in cilia length (Cevik et al., 2013; elongated cilia lengths, associated with an increase in ciliary Craige et al., 2010; Garcia-Gonzalo et al., 2011; Gerhardt et al., 2015). recruitment of IFT88. In addition, our findings reveal novel Thus, we evaluated the role of Ahi1 with regard to ciliary localization of molecular pathways of Arl13b regulation mediated by Ahi1 and the membrane-associated protein Arl13b and on cilia length in Ahi1−/− the involvement of Ahi1 as a centrosome protein important for cells. Wild-type and Ahi1−/− MEFs were serum-starved and migrationprocessesthatprovideinsightintoAhi1dysfunctionin immunolabeled for Arl13b and acetylated α-tubulin (Fig. 2A). A human disease pathogenesis. 25% decrease in ciliary Arl13b intensity was observed in Ahi1-null cells relative to wild type (Fig. 2A,B). Despite the reduction of ciliary RESULTS Arl13b signal in Ahi1−/− MEFs, no changes in intensity were detected Ahi1 localizes at the TZ and its deletion has a small effect on for ciliary acetylated α-tubulin (Fig. 2A,C). Significant reductions in ciliogenesis in MEFs ciliary signal in Ahi1−/− MEFs were also noted for Arl13b:Ac-tub ratios We previously reported that Ahi1 regulates primary cilia formation and measuring Arl13b across a line segmenting cilia (Fig. S2A,B). in epithelial cell lines (Hsiao et al., 2009). Subsequent studies, using Interestingly, cilia length analyses showed significantly longer human-derived fibroblasts from individuals with AHI1 missense cilia in Ahi1−/− MEFs (mean=3.28 µm) compared to wild-type mutations, have shown diverse ciliary phenotypes associated with controls (mean=2.48 µm) (Fig. 2A,D). Exogenous expression of different pathological conditions (Nguyen et al., 2017; Tuz et al., full-length Ahi1–GFP in Ahi1−/− MEFs restored wild-type ciliary 2013). Here, we further explore the involvement of Ahi1 in cilia length (mean=2.70 µm) (Fig. 2D,E) and significantly reversed function, analyzing Ahi1-null MEFs. First, we sought to ciliary Arl13b reductions (∼30% more Arl13b intensity in Ahi1−/− characterize expression and subcellular localization of Ahi1 in cells compared to GFP-transfected Ahi1−/− cells; Fig. 2E,F). These MEFs. Immunoblotting of Ahi1 in MEFs and postnatal brain tissue results confirm the role of Ahi1 in Arl13b ciliary recruitment and in lysates from wild-type and Ahi1−/− mice demonstrate the specificity regulating ciliary length. Notably, the cilia enlargement observed here of our anti-Ahi1 antibody (Fig. 1A). Immunofluorescence analysis in Ahi1−/− MEFs (FVB/NJ) is in contrast to the severe defects in of cells in G0/G1 phase with primary cilia showed Ahi1 localization ciliogenesis previously reported in MEFs lacking Ahi1 from mice on at the base of the ciliary axoneme, colocalized with acetylated a C57BL/6J background (Hsiao et al., 2009) suggesting genetic α-tubulin (Ac-tub) (Fig. 1B). More detailed observations of Ahi1 modifier effects associated with strain background. localization utilizing the basal body marker, γ-tubulin, in addition to If less trafficking of Arl13b to cilia in Ahi1−/− cells is due to Ahi1 and acetylated α-tubulin, revealed that Ahi1 was detected diminished cellular levels of this protein, Arl13b exogenous between the basal body and ciliary axoneme (Fig. 1C), a domain overexpression should reestablish its ciliary translocation. To recognized as the ciliary TZ. The specificity of Ahi1 localization examine this, MEFs were transfected with Arl13b–mCherry and was further confirmed using immunocytochemistry in Ahi1−/− cells cells were immunolabeled with acetylated α-tubulin after primary (Fig. 1B,C). In cells at G2/M transition and S phase, Ahi1 was also cilia induction (Fig. 2G,I). In transfected cells, ciliary Arl13b– detected near and adjacent to centrioles (visualized with γ-tubulin; mCherry intensity was still decreased (∼70%) in Ahi1-null cultures Fig. S1A). In wild-type MEFs treated with nocodazole, Ahi1 is compared to Ahi1+/+ controls (Fig. 2G,I). Interestingly, ectopic detected in proximity to one of the centrioles (mother centriole) expression of a Myc-tagged Arl13b mutant (Arl13b C8S/C9S-myr), (Hsiao et al., 2009), independent of microtubule polymerization which is not palmitoylated but is able to traffic to cilia owing to the (Fig. S1B). These observations demonstrate that Ahi1 is primarily myristoylation (myr) sequence (Roy et al., 2017), is not detected at paired with the mother centriole during the cell cycle, including its primary cilia in Ahi1−/− MEFs (Fig. S2C). These results indicate that localization in proximity to the basal body in ciliated cells (Hsiao deficits in ciliary trafficking of Arl13b in Ahi1-null cells are not et al., 2009; Lee et al., 2014). related to Arl13b palmitoylation. We also examined ciliary Arl13b We then examined ciliogenesis in MEFs assessed by distribution in Ahi1−/− and wild-type embryonic brain tissue, immunofluorescence analysis using the ciliary markers, Arl13b showing a significant reduction in Arl13b-positive cilia in Ahi1−/− and acetylated α-tubulin (Fig. 1D). A modest but significant mice compared to wild-type brains (Fig. S2D). Together, this reduction in Arl13b-positive cilia was observed in Ahi1−/− MEFs supports a role for Ahi1 at the TZ in translocating Arl13b into the grown in 10% FBS (0 h; 10% versus 21% in wild-type cells) or cilium, independent of Arl13b expression levels. serum-starved (48 h; 63% versus 73% in controls) (Fig. 1E). We next assessed ciliary translocation of another ciliary Similarly, a significant reduction in acetylated α-tubulin-positive membrane-associated protein (in neurons), the serotonin 6 receptor cilia was detected in serum-starved (48 h) Ahi1−/− MEFs (∼53% (Htr6), in MEFs (Brailov et al., 2000; Mitchell and Neumaier, 2005). versus 63% in controls) (Fig. S1C) with all acetylated α-tubulin- Heterologous expression of Htr6–EGFP in MEFs showed positive cilia (in both genotypes) colabeling with Arl13b. translocation of this receptor to the primary cilium of transfected In contrast to the substantial reduction in primary cilia wild-type cells (Fig. 2H,I). Consistent with our results obtained for biogenesis previously observed in Ahi1-knockdown mouse inner Arl13b overexpression, a significant reduction in the fluorescence medullary collecting duct cells (IMCD3) and Ahi1−/− MEFs signal for Htr6–EGFP was detected in cilia of Ahi1−/− MEFs from mice on a C57BL6/J background (Hsiao et al., 2009), the compared to wild-type cells (Fig. 2H,I). modest effect on cilia formation observed here in Ahi1−/− MEFs Besides the ciliary deficiency of Arl13b in Ahi1−/− MEFs, we suggests a cell type (organ)-specific effect as well as genetic examined whether ciliary recruitment of platelet-derived growth modifier effects. Differences in cilia phenotypes associated with factor receptor α (Pdgfr-α) in Ahi1-null MEFs. In fibroblasts, Pdgfr-α the cell type (organ) have also been reported in mice mutants is selectively targeted to primary cilia assisting in coordinated cell for other TZ proteins (Garcia-Gonzalo et al., 2011; Weatherbee migration (Schneider et al., 2010, 2005). Our results showed et al., 2009). no significant changes in the ciliary intensity of Pdgfr-α Journal of Cell Science

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Fig. 1. Ahi1 localizes at the ciliary TZ and facilitates cilia formation. (A) Immunoblotting of MEFs and brain tissue lysates from Ahi1+/+ and Ahi1−/− mice probed with Ahi1 and actin antibodies. No Ahi1 bands were detected in Ahi1−/− mice samples using an antibody directed against the C-terminal domain of the Ahi1 protein. (B) Immunofluorescence for Ahi1 (green) and acetylated α-tubulin (Ac-tub; red) with DNA/nuclei (blue) in Ahi1+/+ and Ahi1−/− MEFs. Arrows point to base of cilia where Ahi1 is detected only in Ahi1+/+ MEFs. Scale bar: 5 µm. (C) Ahi1+/+ and Ahi1−/− MEFs serum-starved for 48 h and immunostained for Ahi1 (green), the basal body marker, γ-tubulin (γ-tub; red) and Ac-tub (magenta). Arrows point to the ciliary transition zone where Ahi1 is detected in Ahi1+/+ MEFs. Scale bar: 5 µm. (D) Ahi1+/+ and Ahi1−/− MEFs immunolabeled for the membrane-associated cilia protein Arl13b (red) and Ac-tub (green), after 48 h of serum starvation. Arrowheads indicate Arl13b-positive cilia staining in grayscale images. Scale bar: 30 µm. (E) Percentage of MEFs with Arl13b-positive cilia in cultures grown in 10% FBS (0 h) and serum-starved for 48 h. n=4 per genotype, experiments were done in quadruplicate and at least two fields taken with a 40× magnification objective were considered per experiment (n>400 cells/group). Error bars represent s.e.m. ****P<0.0001; **P<0.01 (χ-squared test). immunolabeling between Ahi1−/− and wild-type cells in serum- (base-to-tip) of IFT88–EYFP upon the addition of compounds that starved cultures (Fig. S3A,B). Moreover, no differences were found upregulate cilia length (Besschetnova et al., 2010). IFT88 is a in cellular Pdgfr-α expression levels (Fig. S3C–E) or in the activation member of the core IFT-B protein complex, which serves as a of the Pdgfr-α signaling pathway (Fig. S3F–H). Overall, these ‘tubulin module’ that binds and transport tubulin within cilia findings suggest that Ahi1 in MEFs selectively disrupts ciliary (Bhogaraju et al., 2013). To examine whether changes in trafficking of Arl13b and Htr6 and regulates ciliary length (a process anterograde IFT components were associated with abnormally that requires proper IFT machinery). elongated cilia in Ahi1-depleted cells, we specifically analyzed expression levels and ciliary distribution of IFT88 in MEFs (Fig. 3; Ahi1 depletion leads to an abnormal distribution of IFT88 and Fig. S4). Immunoblot analysis showed no differences in IFT88 unaltered IFT140 localization in cilia protein levels in whole-cell extracts between wild-type and Ahi1−/− Renal primary cilia in Ift88-null mice are shorter than normal MEFs either in the presence of FBS (0 h) or in serum-starved culture

(Pazour et al., 2000) and have increased anterograde velocities conditions (48 h) (Fig. S4A,B). Following primary cilia induction, Journal of Cell Science

3 RESEARCH ARTICLE Journal of Cell Science (2019) 132, jcs230680. doi:10.1242/jcs.230680

Fig. 2. See next page for legend.

MEFs were immunolabeled for IFT88, acetylated α-tubulin and significantly longer than wild-type cells (Fig. 3A). These γ-tubulin (Fig. 3A–D). IFT88 accumulation was evident at the observations are consistent with an increase in anterograde and proximal region of the axoneme (or TZ) (arrows) as well as at cilia unaltered retrograde (tip-to-base movement) velocities of the IFT tips (asterisks) (Fig. 3A). IFT88 accumulation at cilia tips was system as previously reported (Besschetnova et al., 2010). −/− markedly increased in Ahi1 cells, cells whose cilia are Quantification of the intensity of IFT88 at primary cilia showed a Journal of Cell Science

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Fig. 2. Ahi1 facilitates recruitment of Arl13b and Htr6 to primary cilia and via Gli3 transcriptional activation (Gerhardt et al., 2015). To regulates cilia length. (A) Cilia immunolabeling in serum-starved Ahi1+/+ evaluate cellular Shh transcriptional activity, Ahi1+/+ and Ahi1−/− −/− and Ahi1 MEFs (48 h). Cells were labeled for Arl13b (red) and acetylated MEFs were treated with SAG (Chen et al., 2002) for 24 h and Gli1 α-tubulin (Ac-tub; green), and DNA/nuclei (blue). Scale bar: 5 µm. (B–D) Graphs of all cilia measured for (B) Arl13b and (C) Ac-tub intensities and and Ptch1 mRNA levels were measured using quantitative real-time (D) cilia length from Ahi1+/+ and Ahi1−/− cultures. Ciliary intensities for the two PCR analysis. Upregulation of both mRNAs were found in SAG- cilia markers were normalized to Ahi1+/+ values. n=4/genotype, experiments treated MEFs with the magnitude of the relative expression for Gli1 were carried out in quadruplicate and n>100 cilia/group. (E,F) Ahi1−/− MEFs being higher than Ptch1 (Fig. 4C,D). These differences in relative electroporated with GFP or Ahi1–GFP (green) and serum-starved for 48 h expression can be explained by the fact that Gli1 is not expressed (24 h post-transfection). Cells were analyzed by immunofluorescence for unless Shh is active (Bai et al., 2002). Conversely, Ptch1 is Arl13b (red). Scale bar: 5 µm. Note that in cells expressing Ahi1–GFP, the fusion protein is localized at the base of the primary cilium (arrow), cells display expressed at a baseline level to serve as a Shh signaling receptor normal cilia and ciliary Arl13b labeling is more prominent than in cells with its upregulation after Shh activation functioning as a negative- expressing GFP only. These parameters were quantified and represented as feedback loop for the pathway (Goodrich et al., 1996). Statistical graphs; cilia length in D and Arl13b ciliary intensity in F. Graphs represent data analysis showed a significant reduction in the levels of Gli1 mRNA obtained from Ahi1−/− cell lines (n=3; n>75 cilia). (G,H) Ahi1+/+ and Ahi1−/− in SAG-treated Ahi1−/− MEFs when compared to wild-type MEFs were co-transfected with either (G) GFP and Arl13b–mCherry or (H) cells (Fig. 4C). These results indicate that Ahi1 regulates Shh – Htr6 EGFP and mCherry (green and red, respectively). Cells were serum- transcriptional signaling independently of Smo ciliary recruitment. starved for 48 h (24 h post-transfection) and labeled for acetylated α-tubulin (Ac-tub; magenta) and DNA/nuclei (blue). Scale bars: 20 µm. (I) Graph of Recently, analysis of cells expressing Arl13b with mutations in its Arl13b–mCherry and Htr6–EGFP ciliary intensities normalized to Ahi1+/+ GTPase domain have shown a reduced Shh response, with unaltered values. Bars represent means from n=3 cell lines/genotype (n≥28 cilia/group). Smo enrichment in cilia after Shh activation (Mariani et al., 2016). All error bars represent s.e.m. ****P<0.0001; ***P<0.001; ns, not significant Furthermore, it was shown that Arl13b has the ability to regulate (Mann–Whitney test for B and C, Dunnett’s multiple comparison test for D, Shh signaling downstream of Smo activation (Caspary et al., 2007). – unpaired two-tailed t-tests for F, Mann Whitney test and unpaired two-tailed We propose that reduced ciliary Arl13b in the absence of Ahi1 t-tests, respectively, for Arl13b–mCherry and Htr6–EGFP intensities for I). contributes to the aberrant activation of the Shh pathway. Because there is also a small reduction in cilia biogenesis in the absence of significant increase in Ahi1−/− MEFs (Fig. 3B), and analysis of the Ahi1 in MEFs, it is difficult to determine whether the reduced Shh staining patterns revealed an altered IFT88 ciliary distribution in response is caused also by these defects or by a combination of both Ahi1−/− cells when compared with wild-type cells (Fig. 3C,D). observed phenotypes in Ahi1−/− cells. Notably, we found a higher distribution of IFT88 in a punctate − − pattern in Ahi1 / MEFs (Fig. 3D). This indicates a positive The loss of Ahi1 decreases Arl13b levels in soluble cell correlation between elongated cilia (Fig. 2D) and the ciliary fractions and at the base of the primary cilium concentration of IFT88 in Ahi1−/− MEFs that is independent of the Given the decrease of ciliary Arl13b observed in Ahi1-null cells, we protein levels of IFT88 in the cell (Fig. S4A,B). In order to next examined Arl13b levels by immunoblotting total cell extracts investigate how Ahi1 may be regulating IFT-A subunits, which of MEFs grown in FBS (0 h) or serum-starved for 48 h (Fig. 5A). In control retrograde IFT, we examined endogenous subcellular serum-starved conditions, a significant decrease in Arl13b levels localization of IFT140 (a core IFT-A component) by (∼20%) was found in Ahi1−/− cultures compared to Ahi1+/+ controls immunofluorescence in Ahi1−/− MEFs (Fig. 3E). IFT140 was (Fig. 5B). Conversely, no differences were detected in acetylated predominantly detected at the ciliary base (open arrows) but is also α-tubulin levels in total cell extracts between the wild-type and present at the cilia tip (arrows) (Fig. 3E). No differences in Ahi1−/− samples (Fig. S4C,D). Next, Arl13b levels were assessed in fluorescence intensity of IFT140 were detected either at the base or membrane and soluble fractions from wild-type and Ahi1−/− MEFs cilia tip in Ahi1-depleted cells compared to controls (Fig. 3F,G). grown in serum-starved conditions (48 h). Subcellular fractionation Collectively, analysis of IFT proteins suggest that the absence of followed by immunoblotting revealed that Arl13b was present in Ahi1 preferentially impaired ciliary localization of IFT-B both membrane and soluble cell fractions, using E-cadherin and components, while IFT-A core members were not affected. Gapdh as subcellular compartmentalization markers, respectively (Fig. 5C). The Arl13b signal is more prominent in the fractions Deletion of Ahi1 decreases Shh pathway signaling enriched for the membrane-associated protein E-cadherin, Canonical Shh signaling, which plays an important role in indicating preferential association of Arl13b with cell membranes development and homeostasis, is coupled to the primary cilium (which include ciliary membranes). Supporting this observation, and its activation requires Arl13b and IFT machinery (Huangfu Pdgfr-α, which is compartmentalized almost exclusively to et al., 2003; Liem et al., 2012). Given that Ahi1 deletion impacts fibroblast cilia (Schneider et al., 2005), also co-fractionated with ciliary trafficking of both IFT88 and Arl13b, we hypothesized that E-cadherin, indicating that membrane fractions are enriched in Ahi1−/− cells may have a disruption in the localization of Shh ciliary membrane components (Fig. S3A,C,E). Decreased levels of pathway proteins and therefore in Shh transcriptional signaling. Arl13b were identified in soluble fractions of Ahi1−/− MEFs After primary cilia induction, we activated the Shh pathway with the compared to wild-type cells. Despite this reduction in the cytosol, small molecule Smoothened agonist (SAG) in Ahi1+/+ and Ahi1−/− no changes in Arl13b expression were detected in cell membrane MEFs, and analyzed the trafficking of Smo to the cilium by extracts (Fig. 5D). Visualization of ciliary Arl13b by immunofluorescence (Fig. 4A). Smo enrichment to the cilium was immunofluorescence in cultured cell lines required preservation of only evident upon SAG treatment (right panel, Fig. 4A) and, membrane components necessitating the avoidance of high- surprisingly, no differences were detected in the ciliary intensity of concentrations of detergent post-fixation (Hua and Ferland, 2017; this Shh pathway effector between wild-type and Ahi1−/− MEFs Larkins et al., 2011). Interestingly, when MEFs were treated with an (Fig. 4B). Previous studies have reported that SAG-treated MEFs extraction buffer containing 0.5% Triton X-100 prior to fixation, resulted in a depletion of the ciliary TZ protein, Rpgrip1l, Arl13b was detected at the base of cilia (Fig. 5E). Quantification of suggesting that the regulation of the Shh pathway may also occur Arl13b intensity at the ciliary base revealed an ∼20% reduction in Journal of Cell Science

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Fig. 3. Ahi1 depletion disrupts ciliary distribution of IFT88 while IFT140 is not significantly affected. (A) Serum-starved Ahi1+/+ and Ahi1−/− MEFs (48 h) were fixed and immunolabeled for IFT88 (green), γ-tubulin (γ-tub; red), Ac-tub (magenta), and DNA/nuclei (blue). Arrowheads indicate the proximal region of the axoneme, and asterisks denote cilia tips. Scale bar: 10 µm. (B) Graph of IFT88 intensities at primary cilia with ciliary intensities being normalized to Ahi1+/+ values. n=4 per genotype, experiments were carried out in duplicate (n≥92 cilia/group). (C) IFT88 (green) immunolabeling patterns at primary cilia in Ahi1+/+ and Ahi1−/− MEFs. The base of cilia was identified by γ-tubulin staining (red) with DNA/nuclei (blue). Scale bar: 5 µm. (D) IFT88 distribution at primary cilia represented as stacked bar graphs and expressed as percentages in Ahi1+/+ and Ahi1−/− MEFs. n, number of cilia per group. Graph represents data obtained from n≥3 cell lines per genotype. (E) Serum-starved Ahi1+/+ and Ahi1−/− MEFs (48 h) were immunostained for IFT140 (green) and Ac-tub (red) with DNA/nuclei (blue). Open arrows indicate cilia bases and arrows denote cilia tips. Scale bar: 5 µm. IFT140 intensities at the base (F) and cilia tip (G) in n=3 cell lines per genotype (n>55 cilia). Error – ’ χ bars represent s.e.m. ****P<0.0001; ***P<0.001; ns, not significant (Mann Whitney test for B, F and G and Pearson s -squared test for D). Journal of Cell Science

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Fig. 4. Lack of Ahi1 leads to reduced Shh signaling activation but unaltered ciliary trafficking of Smo. (A) Serum-starved Ahi1+/+ and Ahi1−/− MEFs (24 h) were treated with DMSO (control) or SAG (a Shh activator) for 24 h. Cells were co-immunolabeled for Smo (red) and acetylated α-tubulin (Ac-tub; green) with DNA/nuclei (blue). Smo localization is only seen in cells treated with SAG (arrows). Scale bar: 5 µm. (B) Graph showing Smo intensities at primary cilia after Shh activation by SAG. n>100 cilia per group (n=4/genotype). Relative expression of Gli1 (C) and Ptch1 (D) mRNAs in Ahi1+/+ and Ahi1−/− MEFs after DMSO (control) or SAG treatment for 24 h. Fold change was normalized to Ahi1+/+ cells (set at 1) treated with DMSO after normalization (Rpl13a mRNA). Bars represent means from n≥3 cell lines/genotype and experiments were carried out in duplicate. Error bars represent s.e.m. ****P<0.0001; **P<0.01; *P<0.05; ns, not significant (Mann–Whitney test for B and unpaired two-tailed t-tests for C and D).

Arl13b intensity at the base of the cilium in Ahi1−/− cells compared Ahi1−/− cells, specifically, increased soluble Arl13b levels without wild-type cells (Fig. 5F). These results indicate that Ahi1 appears noticeable changes of the protein in membrane fractions [including not to have a global effect on translocation of Arl13b to cell ciliary membranes (Fig. S5D,E)]. membranes, but modulates Arl13b levels in the cytosol, ciliary base Despite MG132 increasing Arl13b expression in Ahi1−/− cells and ciliary membrane (Fig. 2). (Fig. 5H), this increase is not sufficient to restore Arl13b ciliary As Arl13b is degraded via the proteasomal pathway (Roy et al., recruitment in Ahi1−/− cells (Fig. 5I,J). In addition, proteasome 2017), we evaluated whether pharmacological inhibition of the inhibition accelerated cilia disassembly and increased cilia length proteasome could increase total cell levels of Arl13b and ciliary (Fig. S5A–C). How proteasome inhibition regulates cilia Arl13b in Ahi1−/− cells. Serum-starved Ahi1−/− cultures were formation and axoneme length has been previously reported and treated with MG132 (proteasome inhibitor) for 6 h followed by indicates a functional relationship between negative regulators of immunoblotting and immunofluorescence microscopic analyses cilia biogenesis and their degradation through the ubiquitin (Fig. 5G,I). Ahi1−/− cells treated with MG132 showed a significant proteasome system (Kasahara et al., 2014). Depletion of these increase in total Arl13b levels, but an ∼40% reduction of ciliary negative regulators, which includes the centrosome protein NDE1, Arl13b intensity compared to controls (DMSO, vehicle) (Fig. 5H,I, also increases ciliary length by modulating the IFT system (Kim

J). Subcellular fractionation analysis showed that MG132-treated et al., 2011; Maskey et al., 2015). Despite Arl13b directly Journal of Cell Science

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Fig. 5. See next page for legend. interacting with IFT-B components, this association apparently is However, proteasome inhibition demonstrated a negative not required for Arl13b ciliary localization (Nozaki et al., 2017). In correlation between ciliary length and Arl13b ciliary regard to why decreased Arl13b degradation in the presence of concentration in cells lacking Ahi1. Overall, these findings MG132 significantly impacts its ciliary localization in Ahi1−/− suggest that the absence of TZ proteins, which includes Ahi1, MEFs, our interpretations are limited due to the unknown decrease proteasomal activity at the ciliary base affecting cilia mechanism that regulates Arl13b trafficking to primary cilia. signaling components including Arl13b. Journal of Cell Science

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Fig. 5. Loss of Ahi1 decreases Arl13b levels in soluble cell fractions and characterization of the fluorescent-tagged Arl13b protein remains − − at the ciliary base. (A) Immunoblotting of MEF lysates from Ahi1+/+ and Ahi1 / elusive. Together, these results establish a relationship between the cultures grown in 10% FBS (0 h) and serum-starved for 48 h probed for Arl13b presence of Ahi1 and Arl13b stability. and tubulin. (B) Quantification of chemiluminescent signals normalized to Ahi1+/+ values; tubulin was used as a loading control. n=4/genotype and experiments were performed in duplicate. (C) Arl13b analysis in membrane Ahi1 promotes cell migration and its loss disrupts Pdgfr-α (M) and soluble (S) fractions by immunoblotting in Ahi1+/+ and Ahi1−/− cell mediated chemotaxis cultures. E-cadherin was used as a control for membrane-bound proteins and Previous work has described a role for Arl13b and Ahi1 in neuronal Gapdh as a control for soluble proteins. (D) Quantification of Arl13b signals in migration (Guo et al., 2015; Higginbotham et al., 2012). Here, we n=4 cell lines/genotype, experiments were performed in duplicate. Tubulin was assessed directional migration in Ahi1-null cultures using two used as a loading control for M and S fractions, and bars represent normalization to Ahi1+/+ values. (E) Ahi1+/+ and Ahi1−/− MEFs immunolabeled different approaches: wound-healing assays recorded by live-cell for Arl13b (green) and acetylated α-tubulin (Ac-tub; red) with DNA/nuclei microscopy and transwell assays (Fig. 7). A significant reduction in −/− (blue). Cells were pre-extracted with buffer containing 0.5% Triton X-100 cell wound closure was observed in Ahi1 cultures compared to and fixed with methanol. Scale bar: 3 µm. Arrows highlight Arl13b wild type over a 6 h imaging period (Fig. 7A,B). At 6 h, the wound immunolabeling at the ciliary base. (F) Arl13b intensities at the ciliary base in Ahi1+/+ cultures was nearly closed (80.7%) in comparison to a +/+ normalized to Ahi1 values. (G,I) Immunoblotting and immunofluorescence 61.54% wound closure in Ahi1−/− cells. Similar results were of Arl13b in Ahi1−/− MEFs treated with vehicle (DMSO) or MG132 for 6 h. Prior −/− obtained in sub-confluent MEF monolayers (Fig. S6A,B). No to MG132 treatment, Ahi1 MEFs were serum-starved (48 h) to induce +/+ formation of primary cilia (I). Arl13b, red, arrowheads, and DNA/nuclei, blue. differences in cell proliferation were observed between Ahi1 and −/− Scale bar: 10 µm. (H,J) Quantitative analysis of Arl13b chemiluminescence in Ahi1 MEFs as assessed by analyzing Ki-67 immunoreactivity cells (H) and Arl13b fluorescent signals at primary cilia (J). Experiments were (data not shown). For transwell migration assays, FBS (10%) was carried out in n=3 cell lines in duplicate. Bars represent values normalized to used as a chemoattractant to stimulate migration of MEFs. Migration controls (DMSO/vehicle) and error bars represent s.e.m. ****P<0.0001; towards FBS was significantly reduced (∼20%) in Ahi1−/− cultures – ***P<0.001; **P<0.01; *P<0.05; ns, not significant [Mann Whitney test in B relative to Ahi1+/+ cells (Fig. 7C). Complementary analysis (0 h), D (M), F and J, and unpaired two-tailed t-tests B (48 h), D (S), and H]. performed in previously established Ahi1-knockdown IMCD3 cells (Hsiao et al., 2009) showed a ∼40% reduction in migrating Ahi1 regulates Arl13b stability cells towards FBS (10%) compared to control-scramble cells To further elucidate regulation of Arl13b mediated by Ahi1, we (Fig. S6C–E). examined degradation rates of Arl13b as function of time in Ahi1−/− Arl13b localization during MEF migration was analyzed in MEFs treated with the protein synthesis inhibitor cycloheximide monolayers of confluent Ahi1+/+ and Ahi1−/− cells, 1 h after they (Fig. 6A). Time course analysis of Arl13b levels indicated a were scratched, and immunolabeled for Arl13b. Arl13b localization significantly faster degradation of Arl13b in Ahi1−/− cells relative to was evident at primary cilia (Fig. 7D). Analysis of the number of wild-type controls (Fig. 6A,B). After 6 h of cycloheximide cells with Arl13b-positive cilia facing the wound showed a treatment, there was a non-significant effect on Arl13b levels in significant decrease in Ahi1−/− monolayers compared to Ahi1+/+ wild-type cells, but a 50% decrease of Arl13b in Ahi1−/− cultures controls (Fig. 7D,E). To address concerns over the potential (Fig. 6B). This significant reduction of Arl13b levels in Ahi1−/− participation of non-ciliary Arl13b in migration (Mariani et al., cells treated with cycloheximide was blocked in the presence of 2016), we analyzed chemotaxis in response to SAG stimulation in MG132 (Fig. 6C,D). This result is consistent with the proteasome non-ciliated MEFs (Fig. S7). No differences in chemotactic degradation pathway described for Arl13b (Roy et al., 2017). response were detected between Ahi1−/− and wild-type cells Moreover, a 30% increase in Arl13b levels was observed in suggesting that the reduction in migration is not due to the cycloheximide-treated (6 h) Ahi1−/− cells expressing Ahi1–GFP as demonstrated increased degradation of Arl13b in MEFs compared to controls (GFP-transfected cells) (Fig. 6E,F). To lacking Ahi1. address potential concerns regarding the specificity of Ahi1–GFP In directionally migrating cells, including fibroblasts, the transient transfection and the increased stability of Arl13b in Ahi1−/− centrosome becomes oriented between the nucleus and the cells in the presence of cycloheximide, we also performed the same leading edge facing the movement (Tang and Marshall, 2012). experimental strategy in wild-type MEFs. Our results confirm that the Importantly, Ahi1 localizes at the centrosome at interphase in MEFs augmented levels of Arl13b are not an indirect effect of Ahi1–GFP (Fig. S1A). To investigate centrosome position in migrating Ahi1+/+ overexpression (Fig. 6E,F). Additional results in MEFs transiently and Ahi1−/− MEFs, confluent monolayers were fixed 1 h after the transfected with Ahi1–GFP and treated with either MG132 or introduction of a scratch, and immunolabeled for γ-tubulin and Ahi1 MG132 and cycloheximide indicated that Arl13b levels observed (Fig. 7F,G). In wild-type cultures, 50% of the cells reorientate the after cycloheximide treatment (6 h, Fig. 6F) are the result of Arl13b centrosome towards the scratch-wound in comparison to 30% in stability and not de novo synthesis of the protein (not shown). To Ahi1−/− MEFs (Fig. 7H). This reduction is associated with explore whether stability defects in Arl13b in Ahi1−/− MEFs could be decreased cell migration in Ahi1-null cells (Fig. 7) and possibly rescued by Arl13b–mCherry, serum-deprived Ahi1+/+ and Ahi1−/− implicates Ahi1 in determining the direction of cell movement. We MEFs were transiently co-transfected with Arl13b–mCherry and further tested the ability of Ahi1−/− MEFs to undergo directional GFP constructs and treated with cycloheximide, with cell lysates migration along a gradient of PDGF-AA in a chemotaxis assay. analyzed by immunoblotting (Fig. 6G). Quantitative analysis showed PGDGF-AA is a ligand for the ciliary receptor Pdgfr-α, which that ectopic expression of Arl13b–mCherry was not able to recue controls directed fibroblast migration (Schneider et al., 2010). We Arl13b stability defects in Ahi1−/− MEFs and that Arl13b–mCherry analyzed whole-cell motility behaviors from time lapse movies by stability was not affected in the absence of Ahi1 (Fig. 6H). The latter tracking the movement of individual Ahi1+/+ and Ahi1−/− cells over suggests that overexpressed Arl13b–mCherry has different 12 h (Fig. 8A). Results showed that the percentage of Ahi1−/− MEFs degradation rates than endogenous Arl13b or that overexpression that are able to respond towards the PDGF-AA source were similar per se masks any Arl13b–mCherry stability defects or a combination to wild-type values (∼72% versus 87%, respectively; red tracks, of both. However, this interpretation has a caveat since biochemical Fig. 8A), supporting no alterations in Pdgfr-α signaling activation Journal of Cell Science

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Fig. 6. See next page for legend.

between cells of these two genotypes (Fig. S3). However, Ahi1−/− respectively) (Fig. 8C). Overall, our migration analyses using cells migrated significantly less distance (94.4 µm) than wild-type different assays indicates that Ahi1 modulates directional cell cells (132.0 µm) (Fig. 8B), and moved with a significantly lower migration in fibroblasts downstream of Pdgfr-α, and possibly by velocity to the PDGF-AA source compared to control cells cellular mechanisms that involve centrosome-dependent pathways −/− +/+ (0.15 µm/min versus 0.2 µm/min for Ahi1 and Ahi1 cells, or other cytoskeletal components. Journal of Cell Science

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Fig. 6. Ahi1 increases the stability and protects Arl13b from proteasomal components (Arl13b, Htr6 and IFT88) to cilia. Moreover, a − − degradation. (A) Immunoblotting of Ahi1+/+ and Ahi1 / MEF lysates treated reduction in Arl13b-positive cilia in embryonic brain tissue was with vehicle (DMSO) or cycloheximide (CHX) for 0, 2, 4 or 6 h, and probed for also observed in Ahi1-null mice. However, we found no differences Arl13b. (B) Arl13b fold-change over the CHX time course (n=4/genotype). α (C) Immunoblots of Arl13b from Ahi1+/+ and Ahi1−/− MEF lysates treated with in the ciliary recruitment of IFT140, Pdgfr- and Smo in Ahi1- vehicle (DMSO), CHX, or CHX and MG132 for 6 h. (D) Quantification of Arl13b depleted MEFs, despite changes in transcriptional regulation of fold-change (with or without CHX and/or MG132). n=3/genotype; experiments Gli1. As such, Ahi1 joins the list of TZ proteins implicated in Shh were carried out in duplicate. Bars represent results normalized to control signaling and speculatively in serotonin neurotransmission. − − values (DMSO/vehicle). (E) Immunoblots of Ahi1 / and wild-type MEF lysates Interestingly, conditional deletion of Ahi1 in neuronal cells results – transfected with either GFP or Ahi1 GFP and probed for Arl13b and tubulin. in decreased levels of serotonin in brain tissue as well as At 24 h post-transfection cells were lysed (0 h) or treated with CHX for 6 h. For A, C and E, tubulin was used as a loading control. (F) Quantification of Arl13b concomitant depressive-like behaviors in mice (Ren et al., 2014). chemiluminescent signals. n≥4/genotype; experiments were carried out in Given the involvement of Htr6 in cognition and memory (Mitchell duplicate. Bars represent data normalized to GFP values. (G) Immunoblotting and Neumaier, 2005; Wesołowska, 2010) as well as the association of Ahi1+/+ and Ahi1−/− MEF lysates co-transfected with Arl13b–mCherry and of AHI1 variants in neuropsychiatric and other neurodevelopmental GFP. At 24 h post-transfection, cells were serum-deprived for 48 h and treated disorders, such as autism and schizophrenia (Alvarez Retuerto et al., with CHX for 6 h and probed with the indicated antibodies. (H) Quantification of 2008; Dixon-Salazar et al., 2004; Ferland et al., 2004; Ingason et al., – Arl13b and Arl13b mCherry levels normalized to wild-type values. Tubulin was 2010), future studies are required to elucidate how pathological used as a loading control; similar results were obtained when GFP was used as a loading control (data not shown). Error bars represent s.e.m. ****P<0.0001; alleles in AHI1 alter serotonin signaling pathways and behavior. ***P<0.001; **P<0.01; *P<0.05; ns, not significant [Dunnett’s multiple Despite major progress, the precise molecular mechanism of how comparison test for B and D, unpaired two-tailed t-tests for F and H cells modulate cilia length is not well understood. Mutations or (endogenous) and Mann–Whitney test for H (Arl13b–mCherry)]. absence of ciliary TZ components associated with JBTS result in cilia length differences (Garcia-Gonzalo et al., 2011; Gerhardt et al., DISCUSSION 2015; Srivastava et al., 2017), implicating cilia length dysregulation Human mutations in AHI1 and ARL13B genes, which often result in in human disease. Here, we found that depletion of Ahi1 abnormally non-functional protein products, are linked to JBTS. However, the elongates cilia in MEFs with this phenotype being rescued by molecular relationship between these two JBTS-associated genes transient transfection of wild-type Ahi1–GFP. Ciliary length and their function in the cell and at primary cilia was unclear. regulation requires an equilibrium between assembly and Here, using Ahi1−/− MEFs, we found that the TZ protein, disassembly at the ciliary tip, which relies on IFT machinery Ahi1, (1) promotes ciliary recruitment of membrane-associated and microtubule dynamics (Kozminski et al., 1993; Marshall and and soluble proteins, including Arl13b and IFT88, (2) controls Rosenbaum, 2001; Stephens, 1997). In mice, depletion of the axoneme length, (3) regulates Arl13b stability by preventing its central component of the IFT complex B, IFT88, which transports proteasomal degradation, (4) modulates Shh signaling activation, proteins from the base to the tip of the cilium, results in shorter or and (5) regulates cell migration. As such, our findings contribute to absent cilia and polycystic kidney disease (Davenport et al., 2007; a better understanding of primary cilia function and should aid in Pazour et al., 2000). Conversely, depletion of retrograde IFT understanding the intricate molecular processes of JBTS and other transport, which modulates cargo trafficking from the tip to the base ciliopathies (Fig. 8D). of the cilium, produces an abnormal swollen morphology of this Genomic studies and characterization of protein–protein organelle and altered Shh signaling (Fu et al., 2016; Liem et al., interactions in mammalian cells have revealed the existence of 2012; Qin et al., 2011). Neither of these cilia morphologies were two main TZ modules referred as the NPHP and the MKS complex detected in Ahi1-null cells based on our immunofluorescence (Garcia-Gonzalo and Reiter, 2017). Ahi1 is considered part of the of analysis using different ciliary markers. However, a positive the Meckel syndrome (MKS) TZ module given that it interacts with correlation was observed between elongated cilia, ciliary B9d1 (Dowdle et al., 2011). Recent studies, employing fluorescence concentration of IFT88, and defective Shh signaling activation in recovery after photobleaching and biomolecular fluorescence Ahi1−/− MEFs. Complementary to our findings, an increase in complementation assays, have revealed that there is a large anterograde and unaltered retrograde velocities of IFT88–EYFP immobile fraction of Ahi1 at the base of cilia and Ahi1 interacts were reported upon the addition of compounds that increase cilia with transiting transmembrane and soluble ciliary proteins as well as lengths in IMCD3 cells (Besschetnova et al., 2010). Conversely, our axoneme-associated proteins (Takao et al., 2017). In addition, immunofluorescence analysis in Ahi1-knockout cells for IFT140, a super-resolution imaging has revealed Ahi1 localization to a ring- core component of the IFT-A complex that controls retrograde IFT, shape domain whose diameter corresponds to that of the ciliary showed no differences in IFT140 accumulation at the ciliary base or membrane (Lee et al., 2014). These findings suggest that Ahi1 is a tip compared to wild-type cells. Collectively, analysis of IFT stable component localized in proximity to the peripheral membrane complexes suggests that the absence of Ahi1 preferentially impairs of the ciliary gating zone, which controls the entry of both soluble ciliary trafficking of IFT-B rather than IFT-A proteins (Fig. 8D). and transmembrane proteins to primary cilia. Recently, it has been shown that Arl13b interacts with IFT-B We have previously reported that Ahi1 localizes to the TZ of subunits IFT46 and IFT56 through its C-terminal domain with ciliated photoreceptors and Ahi1 deletion causes abnormal protein presumably no interactions occurring with IFT-A proteins (Cevik trafficking to photoreceptor outer segments leading to retinal et al., 2013; Nozaki et al., 2017). However, Arl13b interaction with degeneration (Westfall et al., 2010). Subsequent observations have IFT-B components appears to have no impact on its ciliary shown a decrease in the number of Arl13b-positive cilia in localization (Higginbotham et al., 2012; Nozaki et al., 2017). fibroblasts obtained from individuals with AHI1 mutations and Here, we found that Ahi1–GFP exogenously expressed in Ahi1-null JBTS (Tuz et al., 2013), suggesting a role for Ahi1 in trafficking of MEFs localizes at the ciliary base, restores cilia length to wild-type ciliary membrane components. Consistent with a role in TZ MEF cilia length, and, importantly, rescues ciliary Arl13b levels. function, here we demonstrated that Ahi1 selectively regulates These observations indicate a negative association between cilia trafficking of membrane-associated components and soluble length and the amount of ciliary Arl13b in Ahi1-null cells. Similar Journal of Cell Science

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Fig. 7. See next page for legend. phenotypes have also been reported in MEFs in the absence of other signals to accelerate axoneme assembly at the expense of ciliary TZ proteins including, Rpgrip1l, Tcntn2 and B9d1, which suggest membrane components; phenotypes observed for other TZ proteins this group of proteins regulates Arl13b ciliary membrane (Garcia-Gonzalo et al., 2011). We propose that the reduced ciliary composition and cilia length (Dowdle et al., 2011; Garcia- levels of Arl13b in the absence of Ahi1 contribute to the abnormal Gonzalo et al., 2011; Gerhardt et al., 2015). Therefore, we activation of the Shh pathway downstream of Smo (Fig. 8D). In hypothesize that the absence of Ahi1 at the ciliary TZ triggers cell agreement with previous reports, reductions in ciliary Arl13b and Journal of Cell Science

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Fig. 7. Ahi1 facilitates cell motility and centrosome re-orientation in remains unclear, but one possibility is that the soluble fraction of − − wound healing assays. (A) Images of Ahi1+/+ and Ahi1 / MEF cultures at Arl13b is more liable to be degraded, while membrane-associated 0, 3 and 6 h after monolayers were scratched. Cell cultures were grown to proteins are protected. We also ruled out that defective Arl13b confluence, scratched and migration recorded with bright-field microscopy in the presence of FBS (10%). Scale bar: 100 µm. (B) Quantitative analysis of translocation in Ahi1-null cells relies on Arl13b palmitoylation wound closure after 3 and 6 h was performed as described in the Materials and processes (Fig. S2C), instead our findings suggest that Ahi1 disrupts Methods. n=3/genotype, experiments in triplicate with at least three different TZ architecture, affecting Arl13b ciliary recruitment. It is possible fields along the wound being analyzed per experiment. (C) Migration analysis that Ahi1 at the TZ protects Arl13b from premature degradation, − − expressed as percentage of Ahi1+/+ and Ahi1 / MEFs towards FBS (10%) thus facilitating its translocation to the ciliary membrane, and this ≥ using transwell inserts. Counting was performed in 10, 20× magnification may explain the reduced levels of the protein in Ahi1-depleted cells fields/experiment. n=3/genotype, experiments were done in triplicate. (D) Ahi1+/+ and Ahi1−/− monolayers, 1 h post-wounding, immunolabeled for at the cytosol. Further investigation is required to examine the Arl13b (red) with DNA/nuclei (blue). Scale bar: 30 µm. Arrows indicate the stability of Arl13b mutants that are not able to localize to cilia in the direction of migration towards the wound and insets show magnified images of absence of Ahi1 to better understand the molecular relationship the boxed region. Arrowheads show Arl13b cilia labeling. (E) Percentage of between Ahi1 and Arl13b proteasome degradation. cells with Arl13b-positive cilia facing towards the scratch in Ahi1+/+ and Ahi1−/− Previous studies have suggested a role of Arl13b and Ahi1 in cell cultures. n=3/genotype; experiments were performed at least in triplicate. +/+ −/− migration (Casalou et al., 2014; Guo et al., 2015; Higginbotham (F) Ahi1 and Ahi1 MEF confluent monolayers were scratched. Cells were et al., 2012), but the precise mechanism remains elusive. Other allowed to recover for 1 h post-wounding, fixed and immunostained for Ahi1 (green), γ-tubulin (red) and DNA/nuclei (blue). Scale bar, 10 µm. Open arrows studies have also shown that cilia coordinate cell migration in depict the direction of the migration towards the wound with the discontinuous fibroblasts through Pdgfr-α ciliary signaling (Schneider et al., white lines marking the wound. (G) Insets show a magnification of the 2010). The cilia-related defects, including the deficits in Arl13b dotted yellow square in F. Ahi1 is only detected in wild-type cells in proximity to function/stability, observed in the absence of Ahi1 led us to explore the centrioles (arrowhead). (H) Percentage of cells with centrosomes that are directional migration in Ahi1-null MEFs by different approaches. +/+ −/− directed toward the wound edge after 1 h in Ahi1 and Ahi1 MEF Our results showed reduced motility with a smaller number of monolayers (n>150 cells/genotype). Error bars represent s.e.m. ****P<0.0001; ***P<0.001; **P<0.01 (unpaired two-tailed t-tests in B and C and χ-squared ciliated cells along the wound edge of scratched monolayers. −/− tests for E and H). Deficits in cell motility were also detected in Ahi1 MEFs by using transwell assays. Interestingly, we also found that a smaller proportion of Ahi1−/− MEFs reorientate the centrosome towards the alterations in Shh signaling have been also noted in cilia from wound. To better delineate ciliary pathways involved in MEF Rpgrip1l−/− MEFs, independent of the activation of Smo (Gerhardt migration in cells lacking Ahi1, we analyzed the chemotaxic et al., 2015). Because there is a small reduction in ciliated Ahi1−/− response to a PDGF-AA gradient in serum-deprived MEF cultures. MEFs, it is difficult to dissect whether the reduced Shh response is Like wild-type cells, Ahi1−/− MEFs were able to migrate towards also caused by the reductions in cilia frequency. In addition to IFT the PDGF-AA gradient. Statistical analysis indicates no significant participation, cilia length regulation requires other intracellular cues, differences in directionality, which is supported by an unaffected such as gene transcription and protein degradation of cilia- Pdgfr-α ciliary recruitment and Pdgfr-α signaling activation in cells associated components, indicating active involvement of other cell lacking Ahi1 (Fig. S3). However, analysis of the parameters organelles (Gibbons et al., 1994; Kasahara et al., 2014; Tang et al., describing cell movement (migrated distance and velocity) 2013). As such, decreased proteasomal activity at the ciliary base confirmed a reduced motility in Ahi1-null cells compared to wild- was associated with abnormal cilia elongation in Rpgrip1l-null type in the presence of PDGF-AA. Overall, these results suggest that MEFs (Gerhardt et al., 2015). However, Rpgrip1l levels at the TZ Ahi1 regulates cell migration downstream Pdgfr-α signaling and were similar between Ahi1+/+ and Ahi1−/− MEFs suggesting possibly through other molecular mechanisms that involve differences in ciliary molecular pathways, which converge in cilia centrosome or ciliary Arl13b-dependent pathways or a length regulation (Fig. S8). combination of both (Fig. 8D). Interestingly, Ahi1 deficiency decreased the amount of Arl13b at the ciliary base and cytosol without noticeable alteration in cell MATERIALS AND METHODS membrane levels. We also observed a reduction of Arl13b stability Animals due to the proteasomal pathway in Ahi1-null cells using Generation of Ahi1-knockout (Ahi1−/−) mice has been described previously −/− pharmacological approaches. Arl13b stability in Ahi1 MEFs (Hsiao et al., 2009). Mice were bred onto an FVB/NJ genetic background was increased by transient transfection of wild-type Ahi1–GFP (Bourgeois and Ferland, 2019) and genotyped by PCR using genomic tail demonstrating that Ahi1 is sufficient for Arl13b stabilization. The DNA as a template. Mice were maintained on a normal 12-h-light–12-h- ciliary phenotype observed in rescue experiments, involving dark cycle with lights off at 19:00 h. All experimental procedures involving overexpression of Arl13b and Htr6, and pharmacological mice were performed under approval from the Institutional Animal Care and inhibition of Arl13b degradation, eliminates defective trafficking Use Committee (IACUC) of the Albany Medical College, in accordance ’ of ciliary Arl13b in Ahi1-null cells as a consequence of reduced with The National Institutes of Health s Guide for the Use and Care of Laboratory Animals. levels of Arl13b in the cytosol. Palmitoylation is generally found to stabilize proteins by preventing ubiquitylation and degradation. The Arl13b mutant Cell culture, transfection, and drug treatments +/+ −/− (C8S/C9S) protein, which disrupt palmitoylation sites within the Wild-type (Ahi1 ) and Ahi1-knockout (Ahi1 ) mouse embryonic fibroblasts (MEFs) were prepared from embryonic day (E)14.5 embryos protein and its localization to cilia membranes, are dramatically − derived from intercrossing Ahi1+/ mice. Following visceral organ removal, degraded via the proteasome. Additionally, ciliary resorption after ’ ∼ tissue was minced with a sterile razor blade in cold Hank s balanced salt heat shock treatment reduces Arl13b protein levels by 50% in the solution. Tissue was incubated with 0.25% trypsin/EDTA (Gibco) for cell, whereas depalmitoylation blockers mitigate degradation of the 30 min at 37°C and passed multiple times through an 18-gauge needle. The protein (Roy et al., 2017). The mechanism by which this post- cell suspensions were transferred to gelatin coated (0.1% v/v in water) 10 cm translational modification protects Arl13b from degradation tissue culture dishes containing DMEM (4500 mg/l glucose, Sigma- Journal of Cell Science

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Fig. 8. Chemotactic response of Ahi1-null cells to a PDGF-AA gradient and a schematic model depicting how Ahi1 mediates ciliary protein composition and cell migration in MEFs. (A) Track plots (12 h) of serum-deprived Ahi1+/+ and Ahi1−/− MEFs in the presence of a PDGF-AA gradient. The starting point for all cell trajectories is set to (x=0, y=0); n represents the number of aggregated trajectories of individual cells. The blue mark in the graphs depict the ‘center of mass’ for all cell end points for each population. Migrated distances (B) and velocities (C) of wild-type and Ahi1−/− MEFs along the PDGF-AA gradient. Error bars represent s.e.m. ****P<0.0001; **P<0.01 (unpaired two-tailed t-tests in B and C). (D) Illustration of an Ahi1 wild-type cell with localization of Arl13b, Htr6 and IFT88 proteins at the primary cilium. In Ahi1-null cells, the levels of these membrane-associated and soluble proteins are reduced in the cilium with concomitant axoneme lengthening. Lacking Ahi1 at the transition zone not only reduces Arl13b ciliary recruitment but also diminishes stability and levels of this protein in the cytosol. Importantly, Shh signaling activation and cell migration processes mediated by Pdgfr-α are also impaired in Ahi1 null cells. Of note, in this model, proteins that retain the ability to access the ciliary compartment in absence of Ahi1 are not represented, which includes IFT140, Smo and Pdgfr-α. Further structural characterization is required to elucidate the Y-link organization in Ahi1-null cells. Journal of Cell Science

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Aldrich), 10% fetal bovine serum (FBS; SH30070, HyClone), 100 units/ml Immunocytochemistry and fluorescence imaging penicillin and 100 mg/ml streptomycin (Gibco). Cells were split upon MEFs (3×104 cells/cm2) were seeded onto gelatin-coated glass coverslips reaching confluency. For experiments, MEFs were used until passage for varying durations and fixed with 4% paraformaldehyde (PFA) for five with cultures maintained at 37°C in a humidified atmosphere containing 18 min at room temperature or in ice-cold methanol for 5 min at −20°C. 5% CO2. Cells were permeabilized in 0.04% Triton X-100, blocked with 1% bovine MEF transfection was performed using an Amaxa Nucleofector II serum albumin (BSA), and incubated with primary antibodies overnight at (Lonza) in accordance with the manufacturer’s instructions using Ingenio 4°C or 1 h at room temperature. Fluorescent-dye-conjugated secondary electroporation solution (Mirus). Briefly, 106 cells were electroporated antibodies were incubated for 1 h at room temperature. All primary and with 1 µg of DNA of the indicated construct and then seeded on secondary antibodies were added in blocking solution and cells were then gelatinized-coverslips. incubated on an orbital shaker. After primary and secondary antibody For rescue experiments, mouse full-length Ahi1 GFP-tagged plasmids incubations, cells were extensively washed with phosphate-buffered saline were electroporated (as described above) in MEFs and then seeded onto (PBS). DNA was labeled with Hoechst 33342 dye (1 µg/ml) and coverslips gelatin-coated plates or coverslips. After electroporation, cells were were mounted using Fluoromount-G (Southern Biotech). To visualize Ahi1 maintained in culture with media containing 10% FBS for 24 h, followed and Arl13b at the base of the primary cilium, cells were pre-extracted for by serum starvation for 48 h to induce robust primary cilia formation. 1 min in an extraction buffer (100 mM PIPES, 1 mM EGTA, 4% PEG800 For all the starvation experiments, media was supplemented with 0.1% FBS. and 0.5% Triton-X-100) followed by methanol fixation. Images were The proteasome inhibitor, MG-132, the protein translation inhibitor, acquired using a Zeiss confocal microscope LSM800 with Airyscan and a cycloheximide (CHX), and nocodazole were from Sigma (474790, 40×/1.4 numerical aperture (NA) objective or a 63×/1.4 NA oil objective. C1988, M1404, respectively). PDGF-AA (R&D Systems) stock solutions Images were processed with Zen black 2.1 or Zen blue lite 2.3 (Zeiss). were prepared at 100 µg/ml in 4 mM HCl as suggested by the manufacturer. Stimulation of MEFs with SAG and quantitative RT-PCR analysis Antibodies and plasmids Wild-type and Ahi1−/− MEFs were plated on coverslips, serum-starved for For immunocytochemistry, primary antibodies were as follows: 1:1000 24 h and treated with 100 nM SAG (Santa Cruz Biotechnology) for an dilution for Ahi1 (Doering et al., 2008), Smo and Pdgfrα (Santa Cruz additional 24 h. Cells were fixed, immunolabeled and analyzed for Smo Biotechnology; sc-166685 and sc-338, respectively), 1:2000 dilution for localization. For quantitative RT-PCR analysis, MEFs were plated on six- γ-tubulin, acetylated α-tubulin (Sigma-Aldrich; T6557 and T6793, well plates (3.5×105 cells/well) (maintained in culture for 24 h) and treated respectively), Arl13b (UC Davis/NIH NeuroMab Facility; N295B/66), with 3 nM SAG (Chen et al., 2002) or vehicle (DMSO) for 24 h after Arl13b, IFT88 and IFT140 (Proteintech; 17711-1-1-AP, 13967-1-AP and plating. Cells were scraped from the well and total RNA was isolated using 17460-I-AP, respectively). Secondary fluorescent antibodies used were: RNeasy Plus Mini Kit 50 (Qiagen) according to the manufacturer’s Alexa Fluor 488 and Alexa Fluor 546 (Life Technologies; A11001, instructions. Isolated RNA was then converted into cDNA using the Verso A21202, A11034 and A11030, A11035 and A11035), and Dylight 649 cDNA synthesis kit (Thermo Fisher Scientific). Expression levels from cell (Jackson ImmunoResearch; 715-495-151), all at 1:2000 dilutions. For lines (n=3, for both genotypes) were analyzed using SYBR Green master western blot analysis, the following primary antibodies were used at 1:2000 mix (Bio-Rad) and gene-specific qPCR primers using a CFX96 Real Time dilutions, Arl13b (Proteintech; 17711-1-AP), E-cadherin (BD Biosciences; PCR machine (Bio-Rad) with reactions performed in triplicate (experiments 610181), α-tubulin (Abcam; ab4074), GFP and m-Cherry (Clonetech; were carried out in duplicate). The expression levels of Gli1 and Ptch1 were 632381 and 632543), 1:5000 for actin (Millipore; MAB1501), and 1:7000 normalized to Rpl13a with the latter serving as the internal control. Primers for Gapdh (Abcam; ab8245). Horseradish peroxidase (HRP)-conjugated for Gli1, Ptch1 and Rpl13a were obtained from Qiagen (QT01551984, secondary antibodies were used at a 1:10,000 dilution (Invitrogen; 32230 QT00149135 and QT00267197, respectively). Melt curve data analyses and 32260). were also performed after each experiment to ensure the amplification of Expression plasmids pEGFPN3-Htr6 and pAGGS-Arl13b-mCherry only one product and the specificity of the primer sequences. vectors were donated by Dr Kirk Mykytyn (Ohio State University) and Dr Kathryn Anderson (Sloan Kettering Institute), respectively. pmCherry-N1 Crude cell membrane isolation and pEGFP-N1 were obtained from Clontech. pcDNA-Ahi1-GFP was 4 2 previously generated in the laboratory (Hsiao et al., 2009). Arl13b C8S/C9S MEFs (3×10 cells/cm ) were grown on gelatinized six-well cell culture was synthesized with an N-terminal myristoylation (myr) sequence and a plates for 24 h and serum-deprived for 48 h. Cells were washed with cold C-terminal myc-tag (Roy et al., 2017), and then cloned in-frame into PBS, scrapped in 150 µl of detergent-free homogenization buffer (250 mM pCDNA3.1. This myr-Arl13b C8S/C9S-myc construct was generated by sucrose, 1 mM EDTA, 1.5 mM MgCl2, 20 mM HEPES pH 7.4) containing Gene Universal. a protease inhibitor cocktail (Roche; 04693159001). Cell suspensions were collected in 1.5 ml tubes, placed on ice for 10 min, followed by three repeated freeze–thaw cycles with liquid nitrogen and ice. Nuclei and Western blotting (immunoblotting) analyses unbroken cells were removed by centrifugation at 800 g for 10 min. The MEFs in six-well cell culture plates were rinsed with cold PBS, scraped off supernatant was collected and centrifuged again at 28,000 g for 30 min in an the plate, and lysed in 150 µl of RIPA buffer supplemented with protease Allegra X-30R centrifuge (Beckman Coulter). The resulting pellet, and phosphatase inhibitors. Cell lysates were then incubated for 20 min on containing membrane (M) proteins, was washed with fractionation buffer a nutating mixer and centrifuged at 16,000 g for 20 min at 4°C. The and the supernatant with soluble (S) proteins was concentrated using supernatant was transferred to a clean tube and protein concentrations were acetone precipitation. M and S fractions were resuspended in 30 µl of RIPA determined with the Advanced Protein Assay Reagent (Cytoskeleton; buffer (150 mM NaCl, 1.0% Triton X-100, 0.5% sodium deoxycholate, ADV01). MEF lysates were mixed with loading buffer, resolved by 10% 0.1% SDS, 5 mM EDTA, and 50 mM Tris-HCl pH 8.0) supplemented SDS-PAGE or 10% TGX stain-free polyacrylamide gels (Bio-Rad; with protease inhibitor cocktail and phosphatase inhibitors (Roche; 161-0183) and transferred to PVDF membranes (Millipore; IPFL00010). 04906837001) and used for western blot analyses. Membranes were incubated in blocking buffer [5% (w/v) nonfat dry milk in Tris-buffered saline with 0.1% Tween (TBST)] for 1 h at room temperature Cell migration assays and analyses followed by overnight incubation of primary antibodies (diluted in blocking MEFs were plated in 12-well cell culture plates and, upon reaching buffer) at 4°C. Membranes were incubated for 1 h at room temperature with confluence (∼48 h), the cell monolayer was scratched using a 200 µl pipette HRP-conjugated secondary antibodies in TBST. Chemiluminescent tip across the bottom of the dish. Cells were washed extensively and allowed detection was performed using the SuperSignal kit (Thermo Fisher to migrate in medium with 10% FBS in a 37°C incubation chamber with 5% Scientific; 34095) and quantified using a ChemiDoc MP imaging system CO2. This live-cell imaging was performed, immediately after the with Image Lab software (Bio-Rad). monolayers were scratched, using a Leica DM IRB microscope at Journal of Cell Science

15 RESEARCH ARTICLE Journal of Cell Science (2019) 132, jcs230680. doi:10.1242/jcs.230680

10× magnification using bright field microscopy. The area of cell migration For all data, we first assessed whether data were normally distributed. For from the initial wound edge was measured in three random areas per well experiments with a small sample size (n=3 or 4), normality was tested using using ImageJ (Schindelin et al., 2012). Wound closure (as a percentage) was the Shapiro-Wilk test. Depending on the distribution, data were analyzed by determined as follows: [(wound area at t0h−wound area at t3h or 6h)/wound Student’s t-test or using the Mann–Whitney test for datasets without a area at t0h]×100. normal distribution. The tests used are indicated in the corresponding figure Transwell assays were performed using modified Boyden chambers. In legend. Prism 7 (v7.0c) was used for all statistical analyses. Statistical brief, cells were serum-starved overnight, trypsinized and resuspended in significance was set to P<0.05. Significance is marked with asterisks in serum-free medium. The lower chamber was filled with 500 µl of DMEM all figures (*P<0.05; **P<0.01; ***P<0.001; ****P<0.0001; ns, not supplemented with either 10% FBS or FBS-free. Cells (4×104 in 200 µl) significant). Red symbols in bar graphs represent individual data points. were added to the culture inserts (8 µm, Costar) and allowed to migrate through the membrane for 6 h at 37°C in a 5% CO2 atmosphere. Cells in the Acknowledgements upper surface of the membrane were gently removed with cotton pads. We wish to thank Dr Joseph Mazurkiewicz for helpful discussions and Ms Julia Migratory cells, attached to the lower side of the membrane, were fixed with Nalwalk for critically reviewing our manuscript. We wish to thank Dr Kirk Mykytyn ice-cold methanol for 5 min at −20°C and stained with Hoechst 33342 dye (Ohio State University) and Dr Kathryn Anderson (Sloan Kettering Institute) for the (1 µg/ml). The number of cells migrating to the bottom of the well were gift of the pEGFPN3-5Ht6 and pAGGS-Arl13b-mCherry vectors, respectively. counted using nuclei staining (with Hoechst) and fluorescent microscopy. Competing interests Images were obtained with a Zeiss AxioImager-Z1 microscope equipped The authors declare no competing or financial interests. with an AxioCam MRm camera and processed with AxioVision Rel. 4.5 software (Zeiss). Author contributions Conceptualization: J.M.-E., R.J.F.; Methodology: J.M.-E., R.J.F.; Validation: J.M.-E.; Chemotaxis assay Formal analysis: J.M.-E., R.J.F.; Investigation: J.M.-E., R.J.F.; Resources: J.M.-E., For chemotaxis experiments, µ-slide chemotaxis chambers (Ibidi) were used R.J.F.; Writing - original draft: J.M.-E.; Writing - review & editing: J.M.-E., R.J.F.; according to the manufacturer’s instructions with slight modifications. In Visualization: J.M.-E., R.J.F.; Supervision: R.J.F.; Project administration: R.J.F.; brief, MEFs (6 µl of a 3×106 cells/ml suspension) were loaded into the Funding acquisition: R.J.F. central channel of the µ-slide chemotaxis chamber that had previously been coated with gelatin (0.1% v/v in water). MEFs were allowed to attach in the Funding This work was supported in part by a grant from National Institutes of Health (NIH/ culture hood for 40 min, transferred to a 37°C incubator with 5% CO2 and maintained in culture for 24 h. Then, cells were serum deprived for 48 h. Of NINDS; R01NS092062 to R.J.F.). Deposited in PMC for release after 12 months. note, chambers were placed in a sterile 10 cm Petri dish with a wet tissue Supplementary information around it to prevent evaporation. Using the manufacturer’s protocol, a Supplementary information available online at PDGF-AA gradient was created in the chambers [C100 (maximum http://jcs.biologists.org/lookup/doi/10.1242/jcs.230680.supplemental concentration)=200 ng/ml]. Cell migration was recorded over 12 h with an image acquired every 10 min on an inverted microscope (10× objective) References having a 37°C incubator and 5% CO2 atmosphere. For trajectory analysis, Alvarez Retuerto, A. I., Cantor, R. M., Gleeson, J. G., Ustaszewska, A., cells were tracked using the manual tracking plug-in for ImageJ, and tracks Schackwitz, W. S., Pennacchio, L. A. and Geschwind, D. H. (2008). were analyzed using the chemotaxis and migration tool plug-in for ImageJ Association of common variants in the Joubert syndrome gene (AHI1) with (Ibidi) software (Schindelin et al., 2012). The software was also used to autism. Hum. Mol. 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For all experiments, results were obtained using at least four separate litters 2009.11.072 with values obtained for Ahi1−/− MEFs being compared to wild-type Bhogaraju, S., Cajanek, L., Fort, C., Blisnick, T., Weber, K., Taschner, M., Mizuno, N., Lamla, S., Bastin, P., Nigg, E. A. et al. (2013). Molecular basis of littermates. All experiments included 2–4 technical replicates. All images in tubulin transport within the cilium by IFT74 and IFT81. Science 341, 1009-1012. the same set of experiments were acquired with identical confocal doi:10.1126/science.1240985 microscope settings with quantification of cilia intensity performed using Bourgeois, J. R. and Ferland, R. J. (2019). Loss of the neurodevelopmental ImageJ (Schindelin et al., 2012). To compare the intensity of Arl13b, Smo, Joubert syndrome causing protein, Ahi1, causes motor and muscle development Pdgfrα and IFT88 localization in the primary cilium of MEFs, integrated delays independent of central nervous system involvement. 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