Wnts regulate planar cell polarity via heterotrimeric G protein and PI3K signaling

Abstract

In the mammalian cochlea, the planar cell polarity (PCP) pathway aligns hair cell orientation along the plane of the sensory epithelium. Concurrently, multiple cell intrinsic planar polarity (referred to as iPCP) modules mediate planar polarization of the hair cell apical cytoskeleton, including the kinocilium and the V-shaped hair bundle essential for mechanotransduction. How PCP and iPCP are coordinated during development and the roles of Wnt ligands in this process remain unresolved. Here we show that genetic blockade of Wnt secretion in the cochlear epithelium resulted in a shortened cochlear duct and misoriented and misshapen hair bundles. Mechanistically, Wnts stimulate Gi activity by regulating the localization of Daple, a guanine nucleotide exchange factor (GEF) for Gαi. In turn, the Gβγ complex signals through phosphoinositide 3-kinase (PI3K) to regulate kinocilium positioning and asymmetric localizations of a subset of core PCP proteins, thereby coordinating PCP and iPCP. Thus, our results identify a putative Wnt/heterotrimeric G protein/PI3K pathway for PCP regulation.

Complementary biosensors reveal different G-protein signaling modes triggered by GPCRs and non-receptor activators

“…On one hand, it was known from in vitro biochemical experiments that GBA motifs have GEF activity on Gαi proteins and that they also promote the dissociation of Gβγ from Gαi-GDP ( Aznar et al, 2015 ; Coleman et al, 2016 ; de Opakua et al, 2017 ; Garcia-Marcos et al, 2011a ; Garcia-Marcos et al, 2010 ; Garcia-Marcos et al, 2009 ; Garcia-Marcos et al, 2012 ; Garcia-Marcos et al, 2011c ; Marivin et al, 2019 ; Maziarz et al, 2018 ). On the other hand, genetic approaches had also established that the GBA motif of some proteins, like GIV and DAPLE, controls G-protein signaling in cells or whole organisms ( Aznar et al, 2015 ; Garcia-Marcos et al, 2011a ; Garcia-Marcos et al, 2010 ; Garcia-Marcos et al, 2009 ; Garcia-Marcos et al, 2012 ; Landin Malt et al, 2020 ; Leyme et al, 2016 ; Leyme et al, 2017 ; Leyme et al, 2015 ; Lin et al, 2014 ; Lo et al, 2015 ; Lopez-Sanchez et al, 2014 ; Ma et al, 2015b ; Marivin et al, 2019 ; Midde et al, 2015 ; Sasaki et al, 2015 ). Thus, an unresolved question so far had been the relative contribution of the two potentially overlapping mechanism of G-protein activation-mediated GBA motifs, that is, generation of Gαi-GTP and formation of free Gβγ.…”

Non-Canonical Wnt Signaling Regulates Cochlear Outgrowth and Planar Cell Polarity via Gsk3β Inhibition

“…On the other hand, we and others have recently demonstrated that secreted Wnts are required for asymmetric localizations of a subset of PCP proteins in inner ear sensory epithelia, including Fzd3/6 and Dvl2 (Landin Malt et al, 2020;Najarro et al, 2020). Importantly, we have shown that asymmetric localization of Fzd6 is controlled by a Wnt/G-protein/PI3K signaling pathway (Landin Malt et al, 2020). In this study, we leverage the inner ear sensory epithelium and genetic tools available to further illuminate the precise relationship between the mammalian non-canonical Wnt and PCP pathways.…”

“…Fzd3/6 are components of the mammalian PCP pathway (Wang et al, 2006;Chang et al, 2016); however, to date, their specific Wnt ligands have not been identified (Sato et al, 2010;Yu et al, 2012;Voloshanenko et al, 2017). On the other hand, we and others have recently demonstrated that secreted Wnts are required for asymmetric localizations of a subset of PCP proteins in inner ear sensory epithelia, including Fzd3/6 and Dvl2 (Landin Malt et al, 2020;Najarro et al, 2020). Importantly, we have shown that asymmetric localization of Fzd6 is controlled by a Wnt/G-protein/PI3K signaling pathway (Landin Malt et al, 2020).…”

“…Thus, kinocilium positioning is crucial for hair bundle polarity and orientation. This process is coordinately controlled by intercellular PCP signaling, several iPCP signaling modules, and a novel, non-canonical Wnt/Gprotein/PI3K signaling pathway (Tarchini and Lu, 2019;Landin Malt et al, 2020). To shed light on the crosstalk and integration of these signaling pathways, we sought to identify cochlear effectors of non-canonical Wnt signaling.…”

Multiple PDZ Domain Protein Maintains Patterning of the Apical Cytoskeleton in Sensory Hair Cells

“…GNAI-GPSM2 is enriched and polarized independently from planar cell polarity (PCP) proteins [6, 8], the machinery that regulates HC orientation via intercellular communication at apical cell-cell junctions [14, 15]. Accordingly, polarization of a subset of PCP proteins was recently proposed to depend on Wnt secretion, but severely disrupted cochlear elongation and morphogenesis in Wntless ( Wls ) mutants left GNAI-GPSM2 enrichment intact at the HC surface [16, 17]. Similarly, polarized segregation of GNAI-GPSM2 and aPKC was preserved in mutants lacking other polarity proteins that occupy the lateral apical HC junction: DAPLE/CCDC88C [18] and PARD3 [19].…”