© 2017. Published by The Company of Biologists Ltd | Journal of Cell Science (2017) 130, 3698-3712 doi:10.1242/jcs.206201 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.