© 2019. Published by The Company of Biologists Ltd | Journal of Cell Science (2019) 132, jcs230680. doi:10.1242/jcs.230680 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