Clustering and Negative Feedback by Endocytosis in Planar Cell Polarity Signaling Is Modulated by Ubiquitinylation of Prickle

Abstract

The core components of the planar cell polarity (PCP) signaling system, including both transmembrane and peripheral membrane associated proteins, form asymmetric complexes that bridge apical intercellular junctions. While these can assemble in either orientation, coordinated cell polarization requires the enrichment of complexes of a given orientation at specific junctions. This might occur by both positive and negative feedback between oppositely oriented complexes, and requires the peripheral membrane associated PCP components. However, the molecular mechanisms underlying feedback are not understood. We find that the E3 ubiquitin ligase complex Cullin1(Cul1)/SkpA/Supernumerary limbs(Slimb) regulates the stability of one of the peripheral membrane components, Prickle (Pk). Excess Pk disrupts PCP feedback and prevents asymmetry. We show that Pk participates in negative feedback by mediating internalization of PCP complexes containing the transmembrane components Van Gogh (Vang) and Flamingo (Fmi), and that internalization is activated by oppositely oriented complexes within clusters. Pk also participates in positive feedback through an unknown mechanism promoting clustering. Our results therefore identify a molecular mechanism underlying generation of asymmetry in PCP signaling.

Control of vertebrate core PCP protein localization and dynamics by Prickle2

“…Additional analysis confirmed that Pk2 knockdown disrupts asymmetry by suppressing the enrichment of Vangl1 at the cortical regions of the cell (supplementary material Fig. S3B) and, together, these observations are consistent with a previously defined role for Pk in the clustering of Vang (Bastock et al, 2003;Cho et al, 2015;Strutt and Strutt, 2007). We conclude that Pk2 promotes cortical Vangl1 concentration, and that an excess of Pk2 promotes further GFP-Vangl1 enrichment that becomes increasingly unrestricted from the ventroposterior cell face.…”

“…The preferential stabilization of Fmi-Vangl pairs at proximal junctions and Fmi-Fz pairs at distal junctions has been suggested to involve an initial upstream bias that is then amplified via both positive-and negative-feedback loops (Amonlirdviman et al, 2005;Tree et al, 2002). These proposed feedback loops that amplify intracellular PCP asymmetry have been shown in Drosophila to involve a network of positive and negative interactions centered on the cytoplasmic core PCP members (Cho et al, 2015;Strutt and Strutt, 2007). In brief, Pk binds and inhibits the membrane localization of Dsh, but Dgo competes with Pk for Dsh binding, promoting the association of Dsh with Fz (Jenny et al, 2005;Tree et al, 2002).…”

“…In addition, Pk mediates the internalization of Vang or FmiVang molecules that are not associated with stable, patterned complexes (Cho et al, 2015). Because of the delicate balance required for asymmetry between these proteins, an intricate system of ubiquitylation and proteosomal degradation is in place to control the protein levels of core PCP components (Cho et al, 2015;Narimatsu et al, 2009;Strutt et al, 2013a,b), and overexpression or loss of function of one core PCP protein in a given cell is generally sufficient to disrupt core PCP asymmetry and result in a loss or reversal of structural polarity (Adler et al, 2000;Bastock et al, 2003;Strutt and Strutt, 2007;Tree et al, 2002).…”

Planar cell polarity in moving cells: think globally, act locally

“…Imaging of the fly wing disc epithelium, for example, has revealed cytoplasmic endosomal vesicles containing Fz and Dvl that are transcytosed along proximodistally oriented apical microtubules (Shimada et al, 2006). Vang-Pk complexes are also internalized (Cho et al, 2015), and both membrane-associated Fz-Dvl and Vang-Pk complexes exhibit a high rate of turnover, particularly in membrane domains where they are not stably localized . Furthermore, an array of endosomal proteins has been identified in screens for planar polarity defects in flies (Carvajal-Gonzalez et al, 2015;Classen et al, 2005;Gault et al, 2012;Hermle et al, 2013;Mottola et al, 2010).…”

“…Furthermore, an array of endosomal proteins has been identified in screens for planar polarity defects in flies (Carvajal-Gonzalez et al, 2015;Classen et al, 2005;Gault et al, 2012;Hermle et al, 2013;Mottola et al, 2010). This polarized membrane trafficking of fly PCP complexes is thought to be part of the mechanism by which PCP protein asymmetry is initially established (Matis et al, 2014) and subsequently amplified (Cho et al, 2015). Endosomal trafficking of PCP proteins is also involved in establishing their asymmetric localization in vertebrate epithelia (Devenport et al, 2011;Giese et al, 2012).…”