Integrin Α8 and Pcdh15 Act As a Complex to Regulate Cilia Biogenesis in Sensory Cells Linda Goodman and Marisa Zallocchi*

RESEARCH ARTICLE Integrin α8 and Pcdh15 act as a complex to regulate cilia biogenesis in sensory cells Linda Goodman and Marisa Zallocchi*

ABSTRACT sensory neuron physiology (hearing/balance, olfaction and vision), The way an organism perceives its surroundings depends on sensory as well as organ development and function (Delmaghani et al., systems and the highly specialized cilia present in the neurosensory 2016; Grati et al., 2015; Jagger et al., 2011; Rachel et al., 2012). cells. Here, we describe the existence of an integrin α8 (Itga8) and Hair cells, the neurosensory cells of the auditory and vestibular protocadherin-15a (Pcdh15a) ciliary complex in neuromast hair cells systems, are the mechanosensors for the perception of sound and in a zebrafish model. Depletion of the complex via downregulation or head movements. Projecting from their apical surface is the hair loss-of-function mutation leads to a dysregulation of cilia biogenesis bundle, which consists of rows of ascending height actin-filled and endocytosis. At the molecular level, removal of the complex stereocilia tethered to a single primary cilium, the kinocilium blocks the access of Rab8a into the cilia as well as normal recruitment (Cosgrove and Zallocchi, 2014). During hair bundle development of ciliary cargo by centriolar satellites. These defects can be reversed the kinocilium is physically connected via extracellular linkages by the introduction of a constitutively active form of Rhoa, suggesting formed between cadherin-23 (Cdh23) and protocadherin-15 that Itga8–Pcdh15a complex mediates its effect through the (Pcdh15), which are essential for proper hair bundle integrity and activation of this small GTPase and probably by the regulation of function (Kazmierczak et al., 2007; Webb et al., 2011). Mutations in actin cytoskeleton dynamics. Our data points to a novel mechanism CDH23 or PCDH15 are associated with Usher syndrome type I and involved in the regulation of sensory cilia development, with the non-syndromic hearing loss in humans (Cosgrove and Zallocchi, corresponding implications for normal sensory function. 2014). Although the kinocilium is important for the establishment of hair bundle polarity (Jones and Chen, 2008), it may also play KEY WORDS: Sensory cilia, Usher syndrome, Pcdh15, Integrin α8 additional roles as a modulator of mechanotransduction activity in immature hair cells as well as a linkage coupling the hair cell bundle INTRODUCTION to components of the extracellular matrix (ECM) (Roberts et al., Non-motile or primary cilia are centriole-derived, microtubule- 1988; Kindt et al., 2012). Recently, it has been shown that mutations based projections present in most metazoan cell types that are in two kinociliary proteins, Cdc14a and Dcdc2, are associated with involved in sensory processes such as mechanosensation, human recessive deafness (Delmaghani et al., 2016; Grati et al., chemosensation and photosensation (Leroux, 2007; Falk et al., 2015). Zebrafish morphants (MOs) for dcdc2 and cdc14a showed 2015). The formation of cilia includes the assembly of the axoneme kinocilium abnormalities with the concomitant defects in hair cell and the directional transport of ciliary proteins by membranous and morphology and function, reinforcing the notion of a direct non-membranous trafficking (Nachury et al., 2007; Westlake et al., involvement of primary cilia in hair cell function (Delmaghani 2011). In recent years significant progress has been made in the et al., 2016; Grati et al., 2015). identification of components involved in vesicular trafficking Integrins are heterodimeric cell surface receptors composed of α necessary for primary ciliogenesis (Leroux, 2007; Pazour and and β subunits that function as adhesion molecules by binding Bloodgood, 2008). Among the proteins identified, Rab8a, a extracellular matrix (ECM) proteins and as receptors by mediating member of the Rab family of small GTPases, plays a critical signal transduction (Müller et al., 1997). In particular, integrin α8 function (Deretic et al., 1995; Nachury et al., 2007). To promote (Itga8) has an obligatory association with the β1 subunit (Itgb1; cilia biogenesis, Rab8 needs to be activated by its guanine Müller et al., 1997) and is selectively incorporated into the apical nucleotide exchange factor, Rabin8 (also known as RAB3IP), membrane of hair cells during development where it is thought to which is targeted to the base of the cilia by GTP-Rab11-positive initiate the assembly of transmembrane complexes necessary for the vesicles (Nachury et al., 2007; Westlake et al., 2011; Hehnly et al., maturation of apical structures (Littlewood-Evans and Müller, 2012). In vertebrates, primary cilia are implicated in several 2000). Itga8-deficient mice have balance problems, abnormal developmental pathways and act as signaling centers mediating stereocilia and fusions between stereocilia and kinocilium, with intercellular communications (Ezratty et al., 2011; Breunig et al., perturbations in the distribution of ECM components. At the 2008; Delling et al., 2016). Dysfunction of cilia, due to mutations in molecular level, Itga8 modulates actin stress fiber assembly via a ciliary proteins or proteins involved in vesicular transport, is Rhoa-dependent mechanism (Zargham et al., 2007a,b; Benoit associated with a broad spectrum of human disorders that affect et al., 2009). Mutations in Itga8 have been associated with bilateral renal agenesis and Fraser syndrome (Humbert et al., 2014; Talbot et al., 2016). Center for Sensory Neuroscience, Boys Town National Research Hospital, Omaha, NE 68131, USA. The present work focuses on the kinocilium of neuromast hair cells. Using zebrafish as the experimental model, we demonstrated *Author for correspondence ([email protected]) ciliary localization and an association between Itga8 and Pcdh15. L.G., 0000-0001-9564-6426; M.Z., 0000-0002-6520-988X Loss of Itga8 or Pcdh15 function leads to a common phenotype, including kinociliary length dysregulation, impairment of

Received 15 May 2017; Accepted 1 September 2017 endocytosis, and Rab8 and centrin mislocalization. These defects Journal of Cell Science

3698 RESEARCH ARTICLE Journal of Cell Science (2017) 130, 3698-3712 doi:10.1242/jcs.206201 can be explained by a reduction in Rhoa activation, since The number of cells harboring a kinocilium was quantified in constitutively active Rhoa is able to rescue these defects in Itga8 MOs and orbiter mutants (Fig. S2A). No significant differences and Pcdh15a knockdown and mutant zebrafish. were observed at 3 dpf. However, at 5 dpf orbiters showed a significant reduction (∼30–40%) in the fraction of cells carrying a RESULTS cilium compared to that in wild-type (WT) animals. Since the Absence of Itga8 or Pcdh15a affects kinocilia elongation number of mature hair cells per neuromast was similar across and/or maintenance treatments, and because we did not observe any increase in hair cell Preliminary results from our laboratory performed in mouse death as judged with a TUNEL assay (Fig. S2B–E), these results auditory hair cells suggested the existence of a functional Itga8– suggest that Pcdh15a and Itga8 proteins are not only required for Pcdh15 complex. To extend these findings in a more suitable model, efficient cilia elongation in neurosensory cells but also for proper we decided to analyze whether defects in Itga8 or Pcdh15a proteins cilia maintenance. resulted in zebrafish hair cell abnormalities. Knockdown zebrafish To confirm the activation state of Rhoa, rhotekin pulldown and for both of these proteins were generated for by the injection of sub- immunofluorescence experiments were performed in MOs and optimal doses of morpholino suspensions into one-cell stage eggs mutants (Fig. 2; Fig. S3). Total lysates from 1–2 dpf larvae were (hereafter denoted MOs), and analyzed at 3 days post fertilization prepared from controls, Itga8 MOs, Pcdh15a MOs and the (dpf). When studying their gross morphology, ∼30% of the MOs corresponding rescued MOs (+cRNA), and Rhoa activation (Itga8 or Pcdh15a) showed pericardial edema and slight body analyzed by pulldown assay (Fig. 2A). We observed a significant curvature (Fig. S1A–E′ and Table S1). Since these defects were not decrease in active Rhoa abundance (GTP-Rhoa, framed area in observed in the pcdh15a zebrafish mutant lines (orbiters, Seiler Fig. 2A–C) when Itga8 or Pcdh15a were knocked down compared et al., 2005) (Fig. S1F–H), they were considered morpholino off- to controls. This deficiency in Rhoa activation was rescued when target effects and excluded from our experiments. Apical hair cell MOs were co-injected with the full-length cRNAs for itga8 or morphology was analyzed in control, MOs and orbiter mutants by pcdh15a, confirming the direct involvement of these proteins in co-staining for phalloidin (a hair cell bundle marker) and acetylated Rhoa regulation. The Rhoa activation state was also evaluated in the tubulin (an axoneme marker) (Fig. 1A–O). Super-resolution orbiter lines at 5 dpf (Fig. 2B). Again, we observed a significant structured illumination microscopy (SR-SIM) analysis showed a decrease in the amount of GTP-Rhoa (Fig. 2B, framed area) when significant reduction of the kinociliary length in the itga8- and comparing the pull down results from mutant and WT animals pcdh15a-deficient animals (Fig. 1B,C,I–L) compared to that in the (Fig. 2F), directly implicating Pcdh15a in Rhoa activation and at the corresponding controls (Fig. 1A,H,K,L). The orbiter mutations not same time confirming the specificity of the Pcdh15a MO only resulted in an average reduction in the kinociliary length phenotype. Finally, total lysates from 1–2 dpf larvae injected with (Fig. 1L) but also in a dysregulation of ciliogenesis in general, as the itga8 MO suspension alone or in combination with the CA rhoa determined by the broader variation of the individual ciliary lengths cRNAs were employed to validate the activation state of Rhoa (Fig. 1M) and by a shift in the distribution of the kinociliary length (Fig. 2C,G). As expected, co-injection of CA Rhoab (Itga8 MO+b) frequencies towards shorter kinocilia (Fig. 1N). The fact that pcdh15a or CA Rhoad (Itga8 MO+d) resulted in a significant increase of MOs and mutants showed similar kinociliary defects demonstrates active Rhoa (GTP-Rhoa, Fig. 2C, framed area) compared to Itga8 the specificity of the MO phenotype. Since Pcdh15 is required for MOs. However, when both CA rhoa cRNAs were injected together proper hair cell mechanotransduction channel activity (Kazmierczak (Itga8 MO+b&d) we observed a modest, although not significant, et al., 2007) and to exclude the possibility that the shortening of the recovery of GTP-Rhoa, more likely due to the strategy employed to kinocilium may be the result of mechanotransduction channel generate these animals (see Materials and Methods). To confirm the impairment, we analyzed kinociliary length in myosin VIIA mutants downregulation of Itga8 in these MOs and in the CA rhoa cRNAs (marinertc320b;Ernestetal.,2000).Theresultsobtainedfromthese co-injected MOs, western blot experiments were run in parallel with animals (Fig. 1L) demonstrated that although mechanotransduction the pulldowns. The results presented in Fig. 2C (Itga8 immunoblot) activity is impaired (Ernest et al., 2000), kinociliary lengthening is demonstrate that GTP-Rhoa was increased despite a reduction in not, suggesting an independency between both processes. Itga8 protein abundance. Overall the data point to the existence of an Kinociliary length was normal when the MOs were co-injected interrelationship between Itga8 and Pcdh15a protein expression/ with the corresponding cRNAs for Itga8 or Pcdh15a (Fig. 1D,E,K,L), function and Rhoa activation. demonstrating the specificity of the morpholino effect. One of the Rhoa localization was assessed in neuromast hair cells through immediate downstream effectors in Itga8-signaling cascade is the immunofluorescence (Fig. S3) with a mouse monoclonal antibody small GTPase Rhoa (Zargham et al., 2007a,b; Benoit et al., 2009). (anti-Rhoa-1) generated and qualified by our laboratory (Fig. S4I–K). Since Rhoa ciliary localization and involvement in primary A significant reduction in the ciliary fluorescent intensity was observed ciliogenesis has been demonstrated (Liu et al., 2007; Pan et al., in the Itga8 MOs compared to that in controls (Fig. S3A,B,K). When 2007; Hernandez-Hernandez et al., 2013), we decided to assess Itga8 MOs were co-injected with the full-length itga8 or CA rhoa whether constitutively active (CA) Rhoa (Schaefer et al., 2014; cRNAs we observed a modest (although not significant) recovery of Farina et al., 2016) was able to rescue the Itga8 MO phenotype. the fluorescence (Fig. S3C–E,K) compared to MOs values. On the The zebrafish genome possesses five rhoa genes, but only rhoab other hand, Pcdh15a MOs showed a significant increase of Rhoa- and rhoad are expressed at early developmental stages (Zhu et al., positive fluorescence that returned to normal values when the MOs 2006, 2008). cRNAs coding for CA rhoab or rhoad (Schaefer were co-injected with the corresponding cRNA (Fig. S3F,G,L). et al., 2014; Farina et al., 2016) were co-injected with the Itga8 Similar results were observed with the orbiter strong animals, that is morpholino suspension and the kinociliary length analyzed in an increase in Rhoa-associated fluorescence, while orbiter weak these animals. We observed rescue of the morphant phenotype animals did not show any significant changes compared to WT when animals were co-injected with CA rhoad only; suggesting animals (Fig. S3H–J,L). The differences between the orbiter lines that Itga8 effect on cilia elongation is mediated by this GTPase may, very likely, be the result of the different mutant alleles

(Fig. 1F,G,K). expressed in these animals (see Materials and Methods). Journal of Cell Science

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Fig. 1. Downregulation or mutations of Itga8 or Pcdh15a inhibits ciliogenesis. (A–J) SR-SIM of 3 dpf (A–G) and 5 dpf (H–J) larvae immunostained for acetylated tubulin (ac tubulin, red) and counterstained with phalloidin (green). Control morpholino-injected animals (A), itga8 (Itga8 MO, B) or pcdh15a (15a MO, C) morphants, morphants co-injected with itga8 (8 MO+cRNA; D) or pcdh15a (15a MO+cRNA; E) cRNAs, Itga8 morphants co-injected with CA rhoab (+Rhoab; F) or rhoad (+Rhoad; G) cRNAs, WT (H), and orbiter (orb) strong (I) and orbiter weak mutants (J) are shown. Scale bars: 4 µm (A–G), 3.5 µm (H–J). (K,L) Quantification analysis of the kinociliary length. For each independent experiment, the average kinociliary length per neuromast was calculated and expressed as mean±s.e.m. (M) Scatter plot of individual kinociliary lengths for WT and orbiter mutants. (N) Frequency distribution analyses of kinociliary length in WT and orbiter mutants. (O) Cartoon of a neuromast (top view) showing the stained structures: hair bundle in green and kinocilia in red. **P<0.01; ***P<0.001; ns, not significant (one-way ANOVA followed by Dunnett’s multiple comparisons test or two-tailed Student’s t-test). At least five independent experiments were performed. The findings suggest that the absence of Itga8 or Pcdh15a protein kinociliary linkage formation and planar cell polarity (Webb et al., function results in Rhoa inactivation and its mislocalization from or 2011; Lelli et al., 2010). Likewise, apical localization of integrin to the cilia. Again, the fact that we observed the same phenotype in α8β1 in immature hair cells has already been described, with Itga8 pcdh15a MOs and mutants, in both pulldown and mutant mice showing abnormalities in hair bundle and kinocilia immunofluorescence experiments, confirms the specificity of the morphologies (Littlewood-Evans and Müller, 2000). Antibodies MO effect. against Itga8 and Pcdh15a (CD1 variants, Maeda et al., 2017) were developed and qualified by our laboratory (Fig. S4A–H′). At 1 dpf Pcdh15a and Itga8 ciliary localization in zebrafish hair cells in the inner ear (Fig. 3A–B′), we observed Pcdh15a and Itga8 Since itga8- and pcdh15a-deficient animals showed kinocilia colocalization in the hair cell precursors (Tanimoto et al., 2011), with abnormalities (Fig. 1), we investigated Itga8 and Pcdh15a Pcdh15a present at the hair cell bundle (asterisk), while Itga8 localized localization in hair cells. Kinocilia localization of Pcdh15 has in both the hair cell bundle (asterisk) at and the cilium (arrowhead). already been reported for murine hair cells as well as its role in Pcdh15a and Itga8 localization was assessed at up to 5 dpf in hair cells Journal of Cell Science

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Fig. 2. Itga8 and Pcdh15a proteins mediate Rhoa activation. Rhoa pulldown assays performed with total lysates from 1–2 dpf MOs (A,C) and 5 dpf pcdh15a mutants (B). (A) Animals were injected with control, specific Itga8 (Itga8 MO) or Pcdh15a (15a MO) MO or with the specific MO and the corresponding cRNA (+cRNA). (C) Itga8 MOs were also co-injected with the cRNAs for CA rhoab (Itga8 MO+b), CA rhoad (Itga8 MO+d) or both (Itga8 MO+b&d). Itga8 immunoblot (IB Itga8) from controls and the treated animals was performed in parallel with the pulldowns to confirm Rhoa activation in the absence of Itga8 protein expression. GTP-Rhoa, active Rhoa, red framed area; tRhoa, total Rhoa; His-Rhoa, recombinant his-tagged Rhoa. White asterisks denote the beads from the pulldown. Note that in some cases GTP-Rhoa is shown as a doublet including the recombinant protein (red asterisks). (D–G) GTP-Rhoa (from the red framed area) abundance was quantified, normalized to tRhoa and expressed as a percentage of that in controls (mean±s.e.m.). *P<0.05; **P<0.01; ***P<0.001; ns, not significant (one-way ANOVA followed by Dunnett’s multiple comparisons test or two-tailed Student’s t-test). At least three independent experiments were performed for each treatment.

(Fig. 3C–D′). SR-SIM showed colocalization of both proteins in the ciliary localization for these two proteins (Fig. S5C–E), but also an hair cell bundle and kinocilia from WT neuromasts, with an increase in ciliary length when co-transfected (Fig. S5F), accumulation towards the tip of the cilia (Fig. 3D,D′,I). Apical corroborating the direct involvement of Itga8 and Pcdh15a in colocalization of both proteins was also detected in the orbiter ciliogenesis. lines (Fig. 3E,H′,J). However, we observed a mislocalization or re-distribution in those lines, with few hair cells showing a Interdependency and interaction between Itga8 and positive hair bundle (3G–H′, asterisks) or cilia immunostaining Pcdh15a (Fig. 3E,E′,G,G′). Instead, we observed a positive signal around Since defects in Itga8 and Pcdh15a proteins resulted in similar the hair cell bundle of what seems to be the cuticular necklace, a ciliary phenotypes and because both proteins colocalize in hair region known to be highly enriched with transport vesicles cells, we decided to analyze whether there was an interdependency (Kachar et al., 1997). These differences in the distribution of in their expression and/or localization. Immunoblots from 3 dpf Itga8 and Pcdh15a in the orbiter lines were frequently observed morpholino controls (Fig. 4A, top blots) showed several bands for within the same animal, demonstrating heterogeneity within the Itga8 between 100–150 kDa corresponding to the predicted mutant phenotype. However, one observation was consistent, molecular mass for the full-length Itga8 (∼118 kDa, GenBank: pcdh15a mutant neuromasts with short kinocilia lacked Itga8 AEU12477.1) and likely representing different degrees of and Pcdh15a ciliary localization (Fig. 3F,F′,H,H′). glycosylation (hereafter denoted the ‘multiplex’, red asterisk). Itga8 and Pcdh15 ciliary localization and regulation was also Smaller bands (∼90 kDa, ∼75 kDa and ∼50 kDa, black asterisks) analyzed in hTERT-RPE transfected cells. We not only observed were also detected. In the case of Pcdh15, several variants have been Journal of Cell Science

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Fig. 3. Itga8 and Pcdh15a localize at the apical aspect of hair cells. Representative images of embryos/larvae immunostained for Pcdh15a (red), Itga8 (green) and acetylated tubulin (blue). Samples were counterstained with phalloidin (gray). (A–B′) Confocal microscopy analyses of a 1 dpf zebrafish inner ear (dorsal view, anterior to the left). (B,B′) Magnification of boxed area in A,A′.(C–H′) SR-SIM of 5 dpf neuromasts. (C–D′) WT. (D,D′) Magnification of boxed area in C,C′.(E–F′) orbiter strong; (G–H′) orbiter weak. Asterisks denote hair bundle immunostaining. The arrowhead in panel B′ denotes cilia immunostaining. (I,J) Cartoons of neuromasts (top views) showing colocalization (yellow) of Itga8 and Pcdh15a. In WT animals (I), both proteins colocalize at the hair bundle in a sub-set of cells and towards the tip of the kinocilia. In mutant animals (J), there is some colocalization at the hair cell bundle but also around it (cuticular plate). Scale bars: 25 µm (A), 8 µm (B), 4 μm (D), 7 µm (C,E–H). Three independent experiments were performed. described in mouse and zebrafish (Ahmed et al., 2006; Maeda et al., specific MOs (Fig. 4A, bottom right blot; Fig. S6), suggesting that 2014; Reiners et al., 2005; Zallocchi et al., 2012a,b). Pcdh15a these variants may lack the morpholino target sequence or, immunoblots from controls (Fig. 4A, bottom blots) showed alternatively, that they may represent non-specific bands. Finally, expression of the full-length protein (Pcdh15a-CD1, ∼200 kDa, when looking at the expression of the reciprocal protein, we red asterisk) and three additional bands (100 kDa, 50 kDa and observed a reduction of the full-length Itga8 (multiplex, red 40 kDa, black asterisks). Itga8 MOs had a reduction of all the Itga8 asterisk), and 90 kDa and 50 kDa variants in the Pcdh15a MOs variants, while Pcdh15a MOs only showed a decrease of the full- (Fig. 4A, top right blot) and a reduction of the Pcdh15a full-length length and the 100 kDa Pcdh15a variants (Fig. 4A; quantification in and 100 kDa variant in the Itga8 MOs (Fig. 4A, bottom left blot). Fig. S6). Surprisingly, rescue with the corresponding full length Re-expression of Pcdh15a resulted in recovery of the Itga8 small cRNA (+cRNA), not only restored expression of the full-length variants but not the full-length protein, while we observed a modest protein but of the small variants (Fig. 4A; Fig. S6), suggesting they recovery of Pcdh15a variants (full length and 100 kDa) in the Itga8 might arise from post-translational modifications. We note that the rescue MOs (Fig. S6). In the case of the orbiter lines, full-length

50 kDa and 40 kDa Pcdh15a variants were not reduced in the Pcdh15a was reduced (Fig. 4B, bottom blot, red asterisk), while an Journal of Cell Science

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Fig. 4. Interdependency of Itga8 and Pcdh15a protein expression and localization in hair cells. (A,B) Representative immunoblots from 3 dpf Pcdh15/Itga8 MOs (A) and 5 dpf pcdh15a mutants (B). (A) Expression of Itga8 and Pcdh15a was analyzed in controls, MOs (Itga8 MOs or 15a MOs) or MOs with the corresponding cRNA (+cRNA). (B) Expression of Itga8 and Pcdh15a was analyzed in WT and orbiter mutants. Red asterisks denote the full-length protein, black asterisks denote putative small protein variants. The blue asterisk denotes an additional variant not found in WT. Membranes were stripped and re-probed for actin as loading control. IB itga8, Itga8 immunoblot; IB 15a, Pcdh15a immunoblot. Three independent experiments were performed. (C–G′) Confocal images of 3 dpf controls (C,C′), MOs [Itga8 MOs or 15a MOs, (D,D′,F,F′) or MOs plus the corresponding cRNA (+cRNA; E,E′, G,G′)]. Larvae were immunostained for Pcdh15a (red), Itga8 (green) and acetylated tubulin (blue) and counterstained with phalloidin (gray). Arrowheads denote Itga8 and Pcdh15a colocalization at the tip of the cilia in controls and +cRNA MOs but not in MOs alone. Top right corner: number of neuromasts showing apical localization for the corresponding protein versus total number of neuromasts inspected. Two independent experiments were performed. Scale bars: 4.5 µm.

distribution (Fig. 4D,D′). Likewise, both proteins were absent from the kinocilium in the Pcdh15a MOs (Fig. 4F,F′). The lack of Pcdh15a ciliary distribution in the specific MOs that at the same time showed a decrease in ciliary length suggests that the full-length and/or the 100 kDa variants are the forms involved in kinocilium elongation. Finally, co-injection with the corresponding full-length cRNAs (Fig. 4E,E′,G,G′) restored ciliary localization of both proteins (arrowheads in merged images). Collectively, the results demonstrate interdependency between Itga8 and Pcdh15a in terms of protein abundance and targeting, pointing to the existence of a putative protein complex in hair cells. The Pcdh15a–Itga8 interaction was assessed by co- immunoprecipitation (co-IP) and by proximity ligation assay (PLA, Wang and Deretic, 2015) (Fig. 5). Reciprocal co-IPs showed an interaction: Itga8 immunoprecipitated the full-length and the 100 kDa variant of Pcdh15a (Fig. 5A, asterisks), while Pcdh15a immunoprecipitated the multiplex corresponding to the full-length Itga8 protein (Fig. 5D, red asterisk) and the ∼90 kDa variant (black asterisk). Co-IP with irrelevant antibodies, normal guinea pig serum (IP NGpS) and normal rabbit serum (IP NRS), confirmed the specificity of the interaction. The direct immunoprecipitations (Fig. 5B,C) showed that we were able to immunoprecipitate some of the corresponding variants for both Itga8 and Pcdh15a, but not all of them; this might be due in part to the inaccessibility of the antibody to the recognition site. PLA experiments (Fig. 5E–I) performed in 5 dpf WT fish (Fig. 5E) confirmed the existence of an Itga8–Pcdh15a complex (red dots) in hair cells. We observed the presence of the complex in the cell body and the apical aspect of hair cells (Fig. 5E, high magnification inset), suggesting that these proteins are being transported together. PLA results employing the orbiter lines (Fig. 5F,G) showed that although some Pcdh15a and Itga8 variants are still present (Figs 3– 4) they are not forming a complex (no red dots, Fig. 5F,G). Fig. 5H shows a representative images of a PLA-negative control in which one of the primary antibodies was omitted. These findings strongly point to an in vivo interdependency additional prominent band (more likely a truncated form, blue between Pcdh15a and Itga8 in hair cells, where reduced expression asterisk) was observed. In contrast to what we found for the Pcdh15a of one of the proteins affects stability and/or targeting of the other. MOs, we did not observe a reduction of the 100 kDa Pcdh15a band Although there were some apparent discrepancies between Pcdh15a (Fig. 4B, bottom blot, black asterisk) nor of the Itga8 variants MOs and mutants (i.e. differences in the Pcdh15a variants that are (Fig. 4B, top blot) in the orbiter lines. being knocked down and presence/absence of Itga8 variants) that Ciliary localization of Itga8 and Pcdh15a was addressed in the may be the result of the different strategies used to reduce Pcdh15a MOs by confocal microscopy analysis (Fig. 4C–G′). Lack of Itga8 function, the final outcome is the same, an obligatory interaction in the Itga8 MOs resulted in loss of Itga8 and Pcdh15a ciliary between Itga8 and Pcdh15a for efficient protein complex Journal of Cell Science

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importantly, Pcdh15a mutants and MOs showed a reduction of the full-length Pcdh15a (and 100 kDa variant in the case of the MOs) and a decrease in Pcdh15a ciliary distribution, which suggests that this is the variant(s) relevant for kinocilia elongation. As for Itga8, although no experiments were performed in Itga8 mutant lines, the fact that we observed the same phenotype in the absence of Itga8–Pcdh15a protein complex and that we were able to rescue those defects with the corresponding cRNAs, strongly points to the specificity of the Itga8 MO phenotype.

Endocytosis is impaired in hair cells from Itga8 and Pcdh15a MOs and mutants A functional role for the Usher proteins in the regulation of vesicular trafficking has been reported by different groups (Tian et al., 2014; Prosser et al., 2008; Yan et al., 2001; Sorusch et al., 2017). More importantly, primary cilia biogenesis has been linked to vesicular trafficking and to actin-based cytoskeleton rearrangements (Molla-Herman et al., 2010; Luo et al., 2012; Ghossoub et al., 2011). Given the reduction in kinociliary length and the decrease in active Rhoa when the Itga8–Pcdh15a complex is not present, we next addressed whether there is a relationship between these defects and the endocytic/recycling pathway. For this purpose, long-term FM1-43 incorporation (Fig. 6A–O) was used as a proxy for hair cell endocytosis (Ogun and Zallocchi, 2014). Downregulation of Itga8 (Fig. 6A,B) or Pcdh15a (Fig. 6H–I) resulted in a significant decrease in FM1-43 incorporation by the hair cells (Fig. 6G,N). Similar to the Pcdh15 MOs, 5 dpf orbiter mutants also showed a significant reduction in dye uptake (orbiter strong ∼85% reduction, orbiter weak ∼60-75% reduction) compared to WTs (Fig. 6K–N). After FM1-43 incubations of up to 90 min, we did not observe any increase in the fluorescence incorporated by the hair cells in MOs and mutants, suggesting that absence of the protein complex results in complete blockage of the endocytic/recycling pathway (Fig. S7). While co-injection with the itga8 cRNA rescued the phenotype (Fig. 6C,G), pcdh15a cRNA co-injected animals did not show any Fig. 5. Itga8 and Pcdh15a interact in hair cells. (A–D) Representative differences in the fluorescence incorporated compared to the – immunoblots of co-IP studies from 3 dpf larvae showing Itga8 Pcdhs15 specific MOs (Fig. 6J,N). Furthermore, no recovery was observed interaction. IP itga8: Itga8 IP; IP NGpS: IP with pre-immune guinea pig serum; IP 15a, Pcdh15a IP; IP NRS, IP with pre-immune rabbit serum; IB 15a, in these animals after 90 min (Fig. S7B,C,H), indicating that the Pcdh15a immunoblot; IB Itga8, Itga8 immunoblot. Asterisks denote specific modest re-expression of Pcdh15a (Fig. 4A, bottom right blot) may bands (red asterisks, full-length protein; black asterisks, small variants). Three not be sufficient to rescue the endocytic defect. Analysis of dye independent experiments were performed. (E–H) Coronal sections of 5 dpf WT uptake in Itga8 MOs co-injected with the CA Rhoa proteins resulted (E,H) orbiter (orb) strong (F) and weak (G) mutants. Positive PLA (red dots) (E in partial but significant recovery (∼50%) of the fluorescence and high magnification inset), demonstrate an in situ Itga8–Pcdh15a incorporated compared to MOs. The fluorescent intensity reached association in WT but not in pcdh15a mutants (F,G). (H) Negative control in normal values (∼100%) when both CA proteins were co-expressed, which one of the primary antibodies (in this case anti-Itga8) was omitted. Sections were immunostained for acetylated tubulin (blue) and counterstained suggesting that they are both involved in the regulation of the with phalloidin (green). (I) Cartoon of a neuromast (lateral view) showing the endocytic pathway in hair cells (Fig. 6D–F,G). The defects in FM1- stained structures: hair bundle in green, kinocilia and cell bodies in blue and the 43 uptake were specific to neuromast hair cells since different cell Itga8–Pcdh15a complex as red dots. Ten animals from two independent types present in the fish skin were able to incorporate the dye in the experiments were inspected. Scale bar: 6 µm (for E–H), 10 µm (inset in E). MO and mutant animals (Fig. S7J–O). localization to the apical aspect of hair cells. When comparing the Absence of the Itga8–Pcdh15a complex activity alters pattern of protein variants expressed by Pcdh15a mutants and MOs, protein cargo transport to the cilium we observed that the abundance of the small variants was Given that previous works demonstrated a role for Rhoa in basal unchanged (50 kDa and 40 kDa variants) in both types of animals body and vesicular trafficking regulation (Pan et al., 2007; (Fig. 4A,B), correlating directly with the presence of positive Bershteyn et al., 2010; Hernandez-Hernandez et al., 2013) and a immunofluorescence at the apical aspect of the neuromast hair cells relationship between Rhoa activity and Rab8a (Braun et al., 2015), (Figs 3–4). Since Pcdh15 apical localization has been addressed by we further characterized the ciliary defect in MOs and mutants by different groups (Kazmierczak et al., 2007; Lelli et al., 2010; Webb analyzing the status of vesicular (Rab8a, Rabin8 and Rab11a/b) and et al., 2011; Zallocchi et al., 2012a,b; Maeda et al., 2014, 2017; basal body/centrosomal proteins (γ-tubulin and centrin). As Ogun and Zallocchi, 2014), it is very likely that the 50 kDa and described previously (Westlake et al., 2011), we observed Rab8a 40 kDa bands are specific Pcdh15a variants still present in the ciliary localization in control animals (Fig. 7A,G,I). Lack of Itga8 or mutants and MOs and not non-specific associations. Most Pcdh15a proteins resulted in a significant reduction of ciliary Rab8a Journal of Cell Science

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Fig. 6. The Itga8–Pcdh15a complex regulates endocytosis/recycling in hair cells. (A–F,H–M) Confocal images of 3 dpf (A–F,H–J) and 5 dpf (K–M) larvae (labeled as in Fig. 1) incubated with FM1-43 for 30 min. Scale bars: 5.5 µm. (G,N) Quantification of total fluorescent intensity per neuromast. At least five independent experiments were performed for each treatment and total fluorescence intensity expressed as a percentage of that in control (mean±s.e.m.). (O) Cartoon of a neuromast (top view) showing the stained structures: hair cells loaded with FM1-43 are in red, and the hair cell bundle in green. **P<0.01; ***P<0.001; ns, not significant (one-way ANOVA followed by Dunnett’s multiple comparisons test or two-tailed Student’s t-test).

(Fig. 7B,E,H,J,AA) while there was an accumulation of Rab8a at the rescued MOs did not show any recovery (Fig. 7U versus V, and base of the cilia. Co-injection of the MOs with the corresponding Fig. 7CC), more likely due to the modest re-expression of the cRNAs showed a significant recovery from the MO phenotype Pcdh15a variants (Fig. 4A). However, since we observed centrin (Fig. 7C,F, AA). When Itga8 MOs were co-injected with the cRNAs defects in both pcdh15a MOs and mutants, this argues in favor of a coding for the CA Rhoa molecules, we only observed full recovery specific morpholino effect. Centrin immunoblots for the different with CA Rhoad (Fig. 7D,AA) suggesting that this signaling treatments did not show any clear correlation between its molecule is the one involved in normal Rab8a ciliary transport. No distribution at the apical aspect of hair cells and its abundance correlation between Rab8a apical localization and protein (Fig. S8B). Similar to the Rab8a results, this may suggest that abundance was observed between the different treatments proper localization of centrin by the Itga8–Pcdh15a complex may (Fig. S8A), suggesting that the lack of ciliary Rab8 might be be independent of its abundance. Alternatively, since our independent of the levels of total Rab8 expression. Because Rab8a immunoblots were performed employing whole-larva lysates, it ciliary localization depends on its centrosomal activation by Rabin8 is also possible that specific differences related to hair cells may (Nachury et al., 2007; Westlake et al., 2011), we assessed Rabin8 have been masked by total centrin (and Rab8a) expression. The distribution in MOs and mutants. Similar to previous reports abnormalities in centrin distribution were not the result of altered (Nachury et al., 2007; Westlake et al., 2011), we observed punctuate basal body maturation or docking. Immunostaining with the basal (vesicular) distribution of Rabin8 at the apical aspect of hair cells body marker γ-tubulin (Fig. S8H–L) did not show any qualitative (Fig. 7K–P). Quantification of the fluorescent intensity (Fig. 7BB) difference between controls and Itga8- or Pcdh15a-deficient did not reveal any significant differences, suggesting that Rabin8 is animals, suggesting that in the absence of the Itga8–Pcdh15a properly targeted to the periciliary region. Consistent with these complex, the cortical actin network involved in basal body results, the Rab11a/b distribution did not show any differences docking is still intact (Pan et al., 2007) and that the defects in between controls and treated animals (Fig. S8C–G). Since centriolar centrin localization are not due to abnormal basal body positioning satellites are also involved in the trafficking of components but are more likely due to centrin transport. necessary for ciliation, we examined the possible contribution of Finally, to address whether the defects observed in Rab8a and the Itga8–Pcdh15a complex in centrin targeting to the centrosome centrin distribution were the result of Itga8–Pcdh15a protein (Nachury et al., 2007). While hair cells from controls showed the complex impairment and not an indirect effect due to defective characteristic centrin punctate distribution corresponding to basal ciliogenesis, we analyzed the distribution of additional cilia- body/centriole localization (Laoukili et al., 2000; Trojan et al., associated proteins. Fig. S8M–V shows that the axoneme- 2008; Bachmann-Gagescu et al., 2015) (Fig. 7Q,W,Y), Itga8- and associated protein IFT54 (also known as TRAF3IP1; Bizet et al., Pcdh15a-deficient animals showed defects in the transport and/or 2015) and the transition zone-associated protein Cc2d2a distribution of centrin (Fig. 7R,U,X,Z,CC). When Itga8 MOs were (Bachmann-Gagescu et al., 2011) have a normal distribution in co-injected with the full-length cRNA (Fig. 7S) or CA rhoab cRNA Itga8 MOs and Pcdh15a mutant animals, suggesting that the Itga8– (Fig. 7T), centrin localization to the centriole/basal body was Pcdh15a complex specifically regulates Rab8a and centrin restored compared to MOs (Fig. 7CC). On the other hand, pcdh15a distribution via a Rhoa signaling pathway. Journal of Cell Science

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Fig. 7. Lack of the Itga8–Pcdh15a complex activity results in ciliary cargo transport impairment. Confocal images and quantitative data (mean±s.e.m.) for Rab8a (A–H), Rabin8 (K–O) and centrin (Q–X) in control, Itga8 MOs, Pcdh15a MOs and orbiter mutants, and in rescued MOs (labeled as in Fig. 1). Scale bars: 6 µm. (I,J,P,Y,Z) Cartoons of neuromasts (top views) showing the corresponding staining: the hair cell bundle is in green, and Rab8a, Rabin8 or centrin is in red. (I) Control neuromast showing Rab8a ciliary staining. (J) Itga8- or Pcdh15a-deficient neuromast showing apical localization of Rab8a but no staining in the kinocilia. (P) Neuromasts showing apical distribution of Rabin8. (Y) Control neuromast showing basal body/transition zone localization of centrin with weak ciliary staining (pink). (Z) Itga8- or Pcdh15a-deficient neuromast showing centrin localization at the basal body/transition zone and also at the hair cell bundle (yellow). (AA) Rab8a graph. The presence of ciliary Rab8a was evaluated for each treatment in five independent experiments and expressed as percentage of that in control. (BB) Rabin8 graph. Neuromast apical fluorescence was quantified for each treatment in three independent experiments and is expressed as percentage of that in control. (CC) Centrin graph. The centrin punctate distribution correlating with the point of insertion of the kinocilium (basal body/centriole) was qualitatively assessed and the results expressed as percentages of that in control. Five independent experiments were performed. *P<0.05; **P<0.01; ***P<0.001; ns, not significant (one-way ANOVA followed by Dunnett’s multiple comparisons test or two-tailed Student’s t-test).

DISCUSSION transport of Rab8a into the cilium and the targeting of centrin to the The way an organism perceives its surrounding environment relies centrosome. on sensory functions. The cilia, as the sensory organelles that project Although a role in ciliogenesis for Pcdh15 was suggested more from the apical surface of neurosensory cells, are involved in key than 15 years ago (Murcia and Woychik, 2001), this is the first work biological processes such as hearing and balance, vision and presenting direct evidence of Pcdh15 involvement in ciliogenesis in olfaction (Jagger et al., 2011; Jones and Chen, 2008; Tian et al., neurosensory cells. In zebrafish hair cells, Pcdh15a (and Itga8) 2014; Jansen et al., 2015; Sorusch et al., 2014). In this work, we showed apical distribution with an accumulation towards the ciliary used zebrafish as an experimental model to demonstrate the tip, far from the hair cell bundle, suggesting additional roles for existence of an Itga8–Pcdh15a protein complex that is involved in Pcdh15a not related to the formation of the kinociliary link kinocilium biogenesis and maintenance in hair cells. Defects in (Kazmierczak et al., 2007). Moreover, since the role of actin Itga8 or Pcdh15a prevent the formation of the complex, leading to a polymerization in ectosome release from the ciliary tip has been decrease in Rhoa-mediated signaling with a concomitant reduction confirmed (Nager et al., 2017), it is appealing to speculate that the in kinociliary length as well as in the number of kinocilia per Itga8–Pcdh15a complex may be involved in this process through the neuromast. Mechanistically, activated Rhoa promotes the vesicular activation of Rhoa. Journal of Cell Science

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Fig. 8. Itga8–Pcdh15a complex function. (A) Under normal conditions, Itga8b1 associates with Pcdh15a and activates Rhoa (1). By regulating acting dynamics, GTP-Rhoa, modulates the Rab8a (2) and centrin (3) ciliary distribution with the concomitant ciliary lengthening. Normal ciliary morphology and signaling lead to proper endocytic activity (4). (B) Absence of the Itga8b1–Pcdh15a complex from the cilia compartment results in Rhoa inactivation (5), leading to an accumulation of inactive Rab8a (6) at the ciliary base and centrin mislocalization (7). Cilia are shorter and endocytic activity (8) is impaired.

Since Itga8 has an obligatory association with the integrin β1 Centriolar satellites are cytoplasmic non-membranous granules subunit (Itgb1) (Müller et al., 1997), and because evidence for involved in the recruitment of cargo to the base of the cilia (Nachury interactions between protocadherins and Itgb1 exists (reviewed in et al., 2007). The observation that centrin distribution is also Yagi, 2008), our findings suggest the there is a ternary complex affected, suggests that activation of Rhoa by the Itga8–Pcdh15a between Itga8 and Pcdh15a that is mediated through Itgb1. The complex plays a more general role in the transport of ciliary presence of ciliary integrins (i.e. Itgb1) and their role in components (i.e. it is acting as regulator of vesicular and non- mechanosensation via ECM interactions has been previously vesicular trafficking). documented, with the mechanical stimuli resulting in extensive The link between the cilium and endocytosis appears to be ciliary signaling and ECM deposition (McGlashan et al., 2006; conserved in many ciliated organisms (Ghossoub et al., 2011; Praetorius et al., 2004; Seeger-Nukpezah and Golemis, 2012). In the Mehta et al., 2014), with components regulating endocytosis also case of neuromasts, they possess a gelatinous matrix structure, the involved in ciliogenesis and ciliary signaling cascades (Luo et al., cupula, anchored to the hair cells by the kinocilia (McHenry and van 2012; Rbaibi et al., 2012; Coon et al., 2012; Clement et al., 2013). Netten, 2007). The presence of the Itga8–Pcdh15a complex along Based on the above information, our data suggest that lack of Itga8– the ciliary membrane in intimate contact with the cupular ECM Pcdh15a complex formation leads to defects in cilia elongation and, poses the question of its putative role as both a modulator of ECM as a result, in an impairment in endocytosis. Thus, by properly protein deposition and as a mechanosensor receptor involved in the modulating ciliary lengthening and maintenance, the Itga8– detection of external cues that, ultimately, will influence fish Pcdh15a complex is indirectly regulating the endocytic pathway. behavior. Although the effects observed in the pcdh15a MOs were specific While evidence exists for the activation of Rhoa by Itga8 (we observed similar defects with the pcdh15a mutants), we were (Zargham et al., 2007a,b; Benoit et al., 2009), the role of Rhoa in unable to rescue endocytosis activity and centrin distribution in the ciliogenesis is still controversial (Pan et al., 2007; Hernandez- MOs. This may be the result of the modest re-expression of Pcdh15a Hernandez et al., 2013). We found that active Rhoa regulates the that was observed upon cRNA co-injection. transport of ciliary cargo necessary for kinocilia formation and Based on our findings and in combination with multiple lines of elongation in neurosensory cells. This is suggested because centrin evidence (Pan et al., 2007; Bershteyn et al., 2010; Hernandez- and Rab8a are mislocalized upon Rhoa inactivation due to Itga8– Hernandez et al., 2013; Nager et al., 2017; Lai et al., 2015; Farina Pcdh15a complex disruption. Rabin8 interacts and activates Rab8a et al., 2016), we propose a model (Fig. 8) in which the Itga8b1– (Nachury et al., 2007; Westlake et al., 2011; Lai et al., 2015) Pcdh15a complex is activated by cupular ECM ligands. Receptor granting GTP-Rab8a access to the cilia compartment. If Rab8a is activation leads to an increased in GTP-Rhoa with its corresponding phosphorylated on Ser111, the Rabin8 interaction does not occur targeting to the ciliary or plasma membrane. Once activated, and, thus, Rab8a remains inactive (Lai et al., 2015). Since membrane bound-Rhoa modulates actin dynamics and, ultimately, impairment of Itga8–Pcdh15a complex formation did not affect the transport of cargo necessary for proper cilia growth and Rabin8 targeting to the pericentriolar recycling endosome (Nachury function. et al., 2007; Westlake et al., 2011), this suggests that the Rabin8– In summary, our study lays the foundation for future work to Rab8a interaction might be disrupted when the complex is absent, uncover the identity of molecules downstream of Rhoa that lead to pointing to a possible role for Rhoa in the regulation of Rab8a Rab8a and centrin ciliary localization, as well as the molecular phosphorylation in hair cells. requirements for Itga8–Pcdh15a complex formation and activation. Journal of Cell Science

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Ciliary defects result in a diverse array of developmental For the generation of a His-tagged itga8 construct containing the T7 abnormalities in humans collectively known as ciliopathies promoter, two rounds of PCRs were performed. The first PCR used forward (Leroux, 2007; Rachel et al., 2012; Sorusch et al., 2014). The primer 5′-CTATAGGGCCCTTCAGCTAGCATGGACTATACCCGCAC TCAC-3′ and reverse primer 5′-TGGTGGTGCGCCCTTGTGGA formation of this novel protein complex in the ciliary membrane of ′ ′ hair cells suggests the presence of a unique mechanism for the TTTTTCT-3 . The second PCR used forward primer: 5 -ATTAA TACGACTCACTATAGGGCCCTTCAGCTAGC-3′ and reverse primer regulation of ciliogenesis in neurosensory cells and poses some 5′-TTTTATTTCAGTGGTGGTGGTGGTGGTGCGC-3′. The final major questions related to the possible function of similar complex fragment was cloned in a TOPO® TA Cloning® Kit (Thermo Scientific) (es) in other sensory systems, with potential implications for human and colonies screened for the construct with the right orientation. itga8 was pathologies. also cloned in the CT-GFP Fusion TOPO® Expression system (Thermo Scientific) with the following pair of primers: forward primer, 5′- MATERIALS AND METHODS ATGGATTACACTCGGACACACAGGGCTGAAG-3′; reverse primer, Fish lines and husbandry 5′-CCGCGTCTGTGGATTTTTCTGCGGTC-3′. Zebrafish (Danio rerio), AB and TL WT strains were grown at 28.5°C under The pEF6/V5-His TOPO® TA Expression system (Thermo Scientific) standard conditions. Animal care and husbandry were overseen by the was used to clone pcdh15a for both generation of cRNA and expression in Institutional Animal Care and Use Committee at Boys Town National cell cultures. Primers designed for this purpose were as follows: forward Research Hospital. Experimental larvae (1–5 dpf, both sexes) were grown in primer, 5′-TGGGTTGGCAGTAGGCATTAAGTGAAGATG-3′; reverse ′ ′ embryo medium (14 mM NaCl, 0.5 mM KCl, 0.25 mM Na2HPO4, primer, 5 -TACATCGTTCTTGTTGTCATATTTAACATCAGGGC-3 . 0.04 mM KH2PO4, 1.3 mM CaCl2, 1 mM MgSO4 and 4 mM NaHCO3, Colonies were screened for the construct with the right orientation. pH 7.4) at 28.5°C with a 10-h-light–14-h-dark cycle. Animals were cryo- For the generation of constitutively active Rhoab and Rhoad, PCR anaesthetized before the initiation of the experiments. The mutant lines products were obtained by using the following pair of primers: forward 5′- orbiterth263b, orbitertc256e and marinertc320b were provided by Teresa ATGGCAGCAATTCGCAAG-3′ and reverse 5′-TCACAGCAGACA Nicolson (Oregon Health and Science University, Portland, OR) and have GCATTTG-3′ for rhoab; forward 5′-ATGGCAGCTATCCGTAAGAAG- been previously described (Seiler et al., 2005; Ernest et al., 2000). The 3′ and reverse 5′-TCATAACAGCAGGCAGC-3′ for rhoad. PCR fragments orbiterth263b line (orbiter strong) harbors a non-sense mutation that results in were cloned with the TOPO® TA Cloning® Kit (Thermo Scientific) and acoustic and vestibular defects, while the orbitertc256e line (orbiter weak) used as a template for site-directed mutagenesis (GeneArt® Site-Directed harbors a missense mutation that results in a weak acoustic response. The Mutagenesis System, Thermo Scientific) of the glycine residue in position marinertc320b line harbors a nonsense mutation in the myosin VIIA gene that 14 to a valine residue. Primers were designed according to manufacturer’s leads to mechanotransduction channel activity impairment. Because instructions. CA rhoab and rhoad were also cloned in the expression vector homozygous animals for the orbiter and mariner mutation can be pAcGFP1-N1 (Clontech) employing the following pair of primers: forward phenotypically screened when they start swimming, we were only able to NheI, 5′-ATGCTAGCATGGCAGCAATTCGCAAG-3′ and reverse XhoI, use them between 4–5 dpf. Otic (O1 and O2), supraorbital (SO3), 5′-TACTCGAGCAGCAGACAGCATTTGTTGC-3′ for rhoab; forward mandibular (M1 and M2), Middle (MI2) and Opercular (OP1) neuromasts NheI, 5′-GTGCTAGCATGGCAGCTATCCGTAAGAAGCTG-3′ and were analyzed in this work. reverse XhoI, 5′-TACTCGAGTAACAGCAGGCAGCCGCT-3′ for rhoad. All constructs were sequence verified by the University of Nebraska Morpholino and cRNA injections Medical Center DNA Sequencing Core Facility (Omaha, NE). Mismatch controls, scrambled and specific MO were designed by and purchased from GeneTools, OR. For the generation of MOs, suboptimal Antibodies doses were injected in one-cell stage eggs. Itga8 MOs were generated by Guinea pig antibodies against an extracellular peptide sequence injection with 1 ng translation-blocking MO (5′-GGACAAACAAG (REFESKPREVGRVYLY) of zebrafish Itga8 were developed under contract AGTGCATGGCTTCA-3′) and 5 ng splice-blocking MO (5′-TAGTGT with Thermo Scientific. Rabbit antibodies against a C-terminal peptide TTATGTGTTTCTGTAGGCC-3′). Pcdh15a MOs were generated by (KNSDRFGCSPDVKYDNKNDV) of Pcdh15a-CD1 (homologous to human injection with 10 ng of translation-blocking MO (5′-CCTCCGCATCTTCA and mouse PCDH15; Seiler et al., 2005; Maeda et al., 2017) were developed CTTAATGCCTA-3′). For the generation of rhoab MOs, a translation- under contract with Proteintech Group. Mouse monoclonal antibodies against a blocking MO (5′-CTTCTTGCGAATTGCTGCCATTTTG-3′) was used at peptide sequence (VADIEVDSKQVELAC) common to both Rhoa proteins 5.7 ng per injection (Zhu et al., 2008). In the case of rhoad MOs, a were developed under contract with GenScript (anti-Rhoa-1). translation-blocking MO (5′-AGCTTCTTACGGATAGCTGCCAT-3′)was Other antibodies used in this study were: mouse monoclonal anti-actin, used at 8 ng per injection. clone C4 (cat. #MAB1501, Millipore), mouse monoclonal anti-pan-centrin, For the rescue experiments, morpholino-resistant cRNAs were generated clone 20H5 (cat. #04-1624, Millipore), mouse monoclonal anti-acetylated as described previously (Ogun and Zallocchi, 2014). Rescue of Itga8 MOs tubulin 6-11B-1 (cat. #T6793, Sigma-Aldrich), mouse monoclonal anti-γ- was performed with 160 pg of itga8 cRNA while 180 pg of pcdh15a cRNA tubulin clone GTU-88 (cat. #T6557, Sigma-Aldrich), chicken anti-GFP (cat. was used for Pcdh15a MOs. For the rescue of Itga8 MOs with CA rhoa #NB100-1614, Novus Biologicals), mouse polyclonal anti-Rabin8 cRNAs, suboptimal doses of 11.25 pg were used (Zhu et al., 2008). When (RAB3IP) (cat. #H00117177-B01P, Novus Biologicals), mouse co-injected together, each CA rhoa cRNA was at a dose of 8.4 pg. monoclonal anti-Rab8a clone 3G1 (cat. #H00004218-M02, Novus Embryos or larvae were maintained in embryo media and life screened at Biologicals), mouse monoclonal anti-6x-His epitope tag HIS.H8 (cat. room temperature using a stereomicroscope (MZ10F; Leica) with a Plan #MA1-21315, Thermo Scientific), rabbit anti-Rab11a/b (cat. #GTX128847, Apochromat 1.0× objective and a magnification of 6.3×. Bright field images Gene Tex), mouse monoclonal anti-Rhoa 26C4 (cat. #sc-418, Santa Cruz were captured with a camera (DFC310 FX; Leica) and the Application Suite Biotechnology), mouse monoclonal anti-V5 epitope (cat. #R960-25, Thermo V4.0.0 acquisition software (Leica). Scientific), rabbit anti-IFT54 (cat. #ARP55316_P050, Aviva Systems Biology) and mouse anti-Cc2d2a (Bachmann-Gagescu et al., 2011). Generation and cloning of Itga8, Pcdh15a, RhoabV14 and RhoadV14 Cell culture and transfections Total RNA was obtained by TRizol extraction of 3 dpf larvae followed by HeLa and HEK293 cells (obtained from Dominic Cosgrove, Boys Town production of cDNA (SuperScript III, First-Strand kit, Thermo Scientific). National Research Hospital, Omaha, NE) were cultured under standard PCRs for itga8 and pcdh15a were performed with the Expand Long Template conditions. Subconfluent cells were dissociated and electroporated with 5– PCR System (Roche), according to the manufacturer’s instructions. PCRs for 10 µg of the corresponding vector and plated onto fibronectin-coated rhoab and rhoad were performed with the FastStart High Fidelity PCR microscope slides at a ratio of 1:10. After 48 h, cells were fixed and prepared

System (Roche), according to manufacturer’s instructions. for immunofluorescence analysis. Journal of Cell Science

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The human telomerase reverse transcriptase retinal pigmented epithelial BAPTA (Thermo Scientific) to disrupt the tip links and thus block FM1-43 (hTERT-RPE) cell line was purchased from the ATCC and grow in DMEM/ entrance through the mechanotransduction channels, and then for 30 to F12 containing 10% fetal bovine serum (FBS) and 0.01 mg/ml hygromycin 90 min with 3 µM of FM1-43FX (Thermo Scientific) in the presence of B. Lipofectamine 2000 (Thermo Scientific) was used to transfect the cells low BAPTA concentrations. After several washes, animals were fixed with with 5–10 µg of Itga8 or Pcdh15a constructs, and after 24 h switched to PFA, counterstained with phalloidin and processed for fluorescence OptiMEM (Thermo Scientific) for 24-48 h to induce ciliogenesis. Cell were analyses. fixed and prepared for immunofluorescence analysis. Proximity ligation assay experiments Immunofluorescence At 5 days post-fertilization larvae were fixed with 4% PFA overnight, rinsed For co-immunostaining of Itga8, Pcdh15a and acetylated tubulin, 3–5 dpf several times with PBSTx 0.1% and mounted in Optical Cutting zebrafish were fixed with 4% paraformaldehyde (PFA) in 1% PBSTw (PBS Temperature compound for cryo-sectioning at 14 µm. PLA was performed plus 1% Tween 20) overnight at 4°C. Samples were rinsed several times by employing the Duolink® In Situ Red Starter Kit Rabbit PLUS (Sigma- with PBSTw and blocked for 1 h at room temperature (RT) with PBSDT Aldrich) and according to manufacturer’s instructions with the following (0.25% Tween-20 and 1% DMSO in PBS) containing 3% bovine serum modifications: coronal sections of 5dpf larvae were blocked with PBSTx albumin (BSA) and 6% FBS. Incubation of primary antibodies (1:500) was containing 2.5% BSA and 2.5% FBS. Primary antibodies (anti-Itga8, anti- done overnight in PBSTw 0.1%. After several washes, samples were Pcdh15a and anti-acetylated tubulin) were diluted 1:200 in the same incubated with the corresponding Alexa-Fluor-conjugated secondary blocking solution. Secondary anti-guinea pig antibody (cat. #A18777, Life antibodies and Alexa-Fluor 488-conjugated phalloidin (Thermo Technologies) was conjugated to the Probemaker MINUS probe as follows: Scientific). Samples were rinsed thoroughly with PBSTw (0.1% Tween antibody was dialyzed against PBS and then conjugated by employing the 20) and incubated for 30 min at RT with 4% PFA in PBS. After several Duolink in situ Probemaker MINUS (Sigma-Aldrich) according to the washes, larvae were mounted for confocal or SR-SIM analysis. manufacturer’s instructions. Sections were incubated with anti-rabbit PLUS For acetylated tubulin, Rab8 and Rab11a/b immunostaining, samples (1:100), anti-guinea pig MINUS (1:30), Alexa-Fluor 405-conjugated anti- were processed as described previously (Ogun and Zallocchi, 2014). mouse-IgG (1:300) and Alexa Fluor 488-conjugated phalloidin (1:300) in For centrin, γ-tubulin and Rabin8 immunostaining, fish were fixed at 4°C blocking solution. Samples were rinsed several times and mounted using for 4 h with 4% PFA, 4% sucrose and 0.01% Tween 20 in PBS and then using ProLong Diamond antifade reagent for confocal analyses. Negative rinsed twice with PBSDT. In the case of γ-tubulin immunostaining, samples controls were produced by omitting one of the primary antibodies (anti-Itga8 were incubated with ice-cold acetone for 6 min at −20°C, washed for 5 min or anti-Pcdh15a). with distilled water (no rocking) and then washed two more times with PBSDT (rocking). Primary antibodies were diluted in PBSDT containing 3% Terminal deoxynucleotidyl transferase dUTP nick end labeling BSA and 6% FBS at a 1:200 dilution, and samples incubated overnight with The TUNEL assay was performed employing the in situ cell Death detection rocking at 4°C. After several washes, larvae were incubated with Alexa Fluor kit, TMR red (cat. #12156792910, Roche) and according to the 594-conjugated anti-mouse-IgG antibody and Alexa-Fluor-488–phalloidin. manufacturer’s instructions. During the final washes, larvae were For Rhoa and IFT54 immunostaining, larvae were fixed with 4% PFA in incubated with Alexa Fluor 488-conjugated phalloidin. As positive PBS overnight at 4°C and rinsed several times with PBSTx (0.1% Triton X- control, 5 dpf WT larvae were incubated for 2 h with cisplatin 500 µM to 100 in PBS). Samples were incubated for 1 h at 37°C in PBS without induce apoptosis. rocking and then with the primary antibodies against Rhoa-1 (1:200) or IFT54 (1:100) in PBSTx 0.1% containing 2.5% BSA and 2.5% FBS. Co-immunoprecipitation experiments Secondary antibodies were Alexa Fluor 594-conjugated anti-mouse-IgG or Co-IP experiments were performed under conditions that allow detection of AlexaFluor 488-conjugated anti-rabbit-IgG. primary and secondary associations according to Ogun and Zallocchi Cc2d2a immunostaining was performed according to Bachmann- (2014) with some modifications. In brief, 30 μl of a 50% protein A– Gagescu et al. (2015). Sepharose CL-4B slurry (Sigma-Aldrich) was incubated overnight with HeLa and HEK293 cells were fixed with 4% PFA for 30 min at processed 7.5 μl of rabbit anti-Pcdh15a, 7.5 μl of guinea pig anti-Itga8, or the as described previously (Ogun and Zallocchi, 2014) but without corresponding pre-immune normal sera (normal rabbit serum was used as a permeabilization. Primary and secondary antibodies were used at 1:500 in specificity control for anti-Pcdh15a antibody, and normal guinea pig serum blocking solution. Untransfected and transfected hTERT-RPE cells were as a specificity control for anti-Itga8 antibody). Three days post-fertilization fixed with 4% PFA, permeabilized for 7 min with PBS plus 0.1% Triton larvae were homogenized in co-IP buffer (10 mM Tris-HCl, pH 7.4, X-100 and prepared for immunofluorescence studies. 150 mM NaCl, 0.5 mM MgCl2, 0.5 mM CaCl2, and 1% Brij 97) containing In all the cases, samples were mounted under coverslips using ProLong protease and phosphatase inhibitors (Thermo Fisher Scientific), and 1-2 mg Gold antifade reagent with or without DAPI. protein used for each co-IP. Zebrafish confocal images were captured at RT using a Zeiss LSM 800 confocal microscope employing the Airyscan function, except for in the Pulldown assay dye-incorporation studies in which the regular confocal function was used. Pulldown experiments (RhoA activation assay, cat. #BK036, Cytoskeleton, Z-stack images were acquired using the 63×, NA 1.4 oil objective at a Inc.) were performed according to the manufacturer’s instructions with 2×zoom, and with a sectioning set automatically to optimal. Z-stack cell the following modification adapted for zebrafish. A total of 20–30 larvae confocal images were acquired using a 40×, NA 1.3 oil objective. (1–5 dpf) were homogenized in 170 µl cell lysis buffer. The pulldown was Zebrafish SR-SIM images were captured at RT using a Zeiss ELYRA PS.1 performed with 15 µl (25 µg) of Rhotekin-RBD protein beads and super resolution microscope. Z-stack images were acquired using a 63×, NA 200–500 µg of protein. Samples were run on a 15% acrylamide gel, 1.4 oil objective, zoom 2×. Sectioning was set automatically to optimal. transferred for 1 h and membranes were blocked for 1 h at RT with 3% milk. Images were acquired and processed with ZEN 2 black edition software Membranes were incubated overnight with the primary antibody anti-Rhoa (Carl Zeiss). SR-SIM images were acquired and processed with the ZEN 2 blue 26C4 (cat. #sc-418, Santa Cruz Biotechnology) at a dilution 1:250 in edition software (Carl Zeiss). Z-stack images are presented as flat Z-projections. TBSTw (0.05% Tween 20). After several washes, the secondary horseradish Only linear adjustments were made to brightness and contrast, and the final peroxidase (HRP)-conjugated anti-mouse-IgG antibody was added at a figures were assembled using Photoshop and Illustrator software (Adobe). dilution 1:3000 for 1 h at RT in TBSTw. Immunoblots were developed using Pierce ECL Plus Western blotting substrate. Total Rhoa Endocytosis experiments immunoblotting was performed with 30–40 µg of protein. The primary Long-term dye incorporation experiments were performed as described antibody dilution was 1:500 and immunoblots were revealed using Pierce previously (Ogun and Zallocchi, 2014). Briefly, zebrafish larvae (3 dpf SuperSignal West Femto. His-Rhoa (5–10 ng, Cytoskeleton, Inc.) was

MOs and 5 dpf mutants) were pre-incubated for 10 min in the presence of included in each immunoblot as a control for specificity. Journal of Cell Science

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Western blotting centriole) was qualitatively assessed and the final results plotted as Embryos or larvae were deyolked and lysates processed as previously percentages of that in controls. For both immunostainings, at least five described (Ogun and Zallocchi, 2014). Protein samples were loaded at 15– independent experiments were performed with five to ten animals per 35 µg per lane and transferred overnight. For Itga8 and Pcdh15a western treatment and at least one neuromast analyzed per animal. blots, membranes were incubated in 5% milk blocking solution (0.1% To minimize bias during data processing, animal treatments were Tween 20, 75 mM NaCl, 5% glycerol, and 5% non-fat dried milk in PBS) unknown to the person conducting the analyses. overnight. Primary antibody (1:1000) was added in blocking solution overnight with rocking. Membranes were rinsed and incubated with goat Statistical analysis HRP-conjugated anti-rabbit-IgG (1:20,000) or goat HRP-conjugated anti- Numerical data were graphically represented as scattered plots (individual guinea-pig-IgG (1:10,000) secondary antibodies for regular western blots or values) or as bar graphs (means±s.e.m.). Statistical analysis was performed with HRP-conjugated protein A (1:10,000; GE Healthcare) for co-IP using Prism 5 (version 6.07). A non-parametric Kruskal–Wallis test experiments. Membranes were developed using Pierce ECL Western followed by Dunn’s multiple comparisons test was used to compare the blotting substrate (Thermo Fisher). proportion of ciliated hair cells. A two-tailed Student’s t-test was used for For Rhoa immunoblots, employing the antibody developed by our the long-term endocytosis experiments (30–90 min) to compare Itga8 MOs laboratory (anit-Rhoa-1), membranes were incubated for 1 h at RT in 3% versus controls and in all other experiments to compared Pcdh15a MO+ milk blocking solution and the primary antibody overnight at a dilution cRNA versus Pcdh15a MOs. One-way ANOVA followed by Dunnett’s 1:200 in TBSTw 0.05%. Secondary antibody was used at 1:3000 in TBSTw multiple comparisons test were used to analyze the rest of the numerical 0.05%. For Rab8a and centrin-1, immunoblots membranes were blocked for data. Treatments were compared to control (statistics in black) and versus the 1 h at RT with 3% milk blocking solution and primary antibody diluted corresponding MO (statistics in red). to the concentration suggested by the manufacturer. For pan-actin immunoblotting, membranes were stripped with western blot stripping Acknowledgements buffer (Restore PLUS; Thermo Scientific) and probed as previously We thank J. Taylor and J. Talaska for providing assistance with microscopy. Support described (Ogun and Zallocchi, 2014). for the UNMC Advanced Microscopy Core Facility was provided by the Nebraska hTERT-RPE cells were processed as described previously for other cell Research Initiative, the Fred and Pamela Buffett Cancer Center Support Grant types (Zallocchi et al., 2012a,b), and 30 µg of protein used for western blot (P30CA036727), an Institutional Development Award (IDeA) from the NIGMS of the studies. Membranes were stripped and re-probed for actin as a loading NIH (P30GM106397), the Nebraska Research Initiative Grant and Nebraska Center for Cellular Signaling CoBRE (NIH P30GM106397). We thank Dr T. Nicolson control. (Oregon Health and Science University, Portland, OR) for the mutant zebrafish lines, In all the cases, membranes were exposed to films and developed using a Dr S. Rocha-Sanchez (Creighton University) for cisplatin, R. Bachmann-Gagescu film processor. (University of Zurich, Switzerland) for the Cc2d2a antibody, O. Ogun for fish husbandry, D. Delimont for genotyping and S. Kennedy for figure preparation and art Sample size and data analysis work. We also want to thank Dr D. Cosgrove (BTNRH) for critically reviewing the Ciliary lengths were measured by employing ZEN 2 black edition software manuscript. (Carl Zeiss), averaged for each treatment and presented as bar graphs. Individual kinociliary lengths are presented as scattered plot graphs. At least Competing interests five independent experiments were performed; each containing at least ten The authors declare no competing or financial interests. fish and at least two neuromasts per fish were inspected. For hTERT-RPE cells, three independent experiments were performed. Ciliary length was Author contributions Conceptualization: M.Z.; Methodology: L.G., M.Z.; Validation: L.G., M.Z.; Formal measured in control cells and in transfected cells positive for ciliary analysis: M.Z.; Investigation: L.G., M.Z.; Resources: M.Z.; Writing - original draft: Pcdh15a, Itga8 or both. M.Z.; Writing - review & editing: M.Z.; Visualization: M.Z.; Supervision: M.Z.; Project Three independent experiments were performed for the pulldown administration: M.Z.; Funding acquisition: M.Z. assays, co-IPs and the MOs/mutant immunoblots. Each lane in the blots corresponds to 15–60 larvae. Protein bands were quantified by using Funding ImageJ software, version 1.51 g (NIH), and normalized to the total This work was supported by National Institutes of Health (grant 5P20RR018788) amount of Rhoa (tRhoa) (for pull downs) or actin (for regular and the Tobacco Settlement Fund from the State of Nebraska to M.Z. Deposited in immunoblots) and the numerical data expressed as a percentage of that PMC for release after 12 months. in controls. Two independent experiments were performed in the case of the controls, Supplementary information MOs, and rescued MOs immunostained for Itga8 and Pcdh15a. The number Supplementary information available online at of neuromast hair cells showing Itga8 or Pcdh15a apical localization was http://jcs.biologists.org/lookup/doi/10.1242/jcs.206201.supplemental expressed versus the total number of neuromasts inspected. References Two PLA independent experiments were performed with ten animals Ahmed, Z. M., Goodyear, R., Riazuddin, S., Lagziel, A., Legan, P. K., Behra, M., each, and one neuromast per animal was analyzed for the presence or Burgess, S. M., Lilley, K. S., Wilcox, E. R., Riazzudin, S. et al. (2006). The tip- absence of PLA staining. link antigen, a protein associated with the transduction complex of sensory hair Fluorescence intensities were calculated as described in Ogun and cells, is protocadherin-15. J. Neurosci. 26, 7022-7034. Zallocchi (2014) employing ImageJ software, and expressed as percentages Bachmann-Gagescu, R., Phelps, I. G., Stearns, G., Link, B. A., Brockerhoff, of that in controls. For Rhoa and Rabin8 immunostaining, a region of S. E., Moens, C. B. and Doherty, D. (2011). The ciliopathy gene cc2d2a controls interest (ROI) corresponding to the apical region of neuromasts hair cells zebrafish photoreceptor outer segment development through a role in Rab8- was selected and the fluorescence calculated in at least three independent dependent vesicle trafficking. Hum. Mol. Genet. 20, 4041-4055. experiments. At least ten animals were analyzed per treatment and at least Bachmann-Gagescu, R., Dona, M., Hetterschijt, L., Tonnaer, E., Peters, T., de two neuromasts per animal. In the case of endocytosis experiments, the ROI Vrieze, E., Mans, D. A., van Beersum, S. E. C., Phelps, I. G., Arts, H. H. et al. corresponding to the whole neuromast was selected and the total (2015). The ciliopathy protein CC2D2A associates with NINL and functions in fluorescence intensity calculated and plotted as a percentage of that in RAB8-MICAL3-regulated vesicle trafficking. PLoS Genet. 11, e1005575. Benoit, Y. D., Lussier, C., Ducharme, P.-A., Sivret, S., Schnapp, L. M., Basora, control. Five independent experiments were performed, with five to ten N. and Beaulieu, J.-F. (2009). Integrin alpha8beta1 regulates adhesion, larvae per treatment and one or two neuromasts inspected per animal. migration and proliferation of human intestinal crypt cells via a predominant In the case of Rab8a immunostaining, its ciliary localization was RhoA/ROCK-dependent mechanism. Biol. Cell 101, 695-708. qualitatively assessed, and the data expressed as a percentage of that in Bershteyn, M., Atwood, S., Woo, W.-M., Li, M. and Oro, A. E. (2010). MIM and controls. For centrin immunostaining experiments, the punctate distribution cortactin antagonism regulates ciliogenesis and hedgehog signaling. Dev. Cell that correlates with the point of insertion of the kinocilium (basal body/ 19, 270-283. Journal of Cell Science

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