Kermit interacts with Gαo, Vang, and motor proteins in Drosophila planar cell polarity

Abstract

In addition to the ubiquitous apical-basal polarity, epithelial cells are often polarized within the plane of the tissue--the phenomenon known as planar cell polarity (PCP). In Drosophila, manifestations of PCP are visible in the eye, wing, and cuticle. Several components of the PCP signaling have been characterized in flies and vertebrates, including the heterotrimeric Go protein. However, Go signaling partners in PCP remain largely unknown. Using a genetic screen we uncover Kermit, previously implicated in G protein and PCP signaling, as a novel binding partner of Go. Through pull-down and genetic interaction studies, we find that Kermit interacts with Go and another PCP component Vang, known to undergo intracellular relocalization during PCP establishment. We further demonstrate that the activity of Kermit in PCP differentially relies on the motor proteins: the microtubule-based dynein and kinesin motors and the actin-based myosin VI. Our results place Kermit as a potential transducer of Go, linking Vang with motor proteins for its delivery to dedicated cellular compartments during PCP establishment.

Figure 1. Kermit is identified as a novel binding partner of Gαo.

Overexpression of Gαo in Drosophila wings leads to folded wings (A) and multiple hair cells (B). The magenta frame on the wild-type wing in (A) indicates the region magnified in (B-D). The multiple hair phenotype is strongly suppressed in a kermit heterozygous mutant (C) or RNAi against kermit (D) background. Lower panels in (B, C, D) show higher magnification of the selected regions of the wings. (E) Quantification of the multiple hair cells induced by Gαo overexpression by the MS1096-Gal4 driver in different genotypes. Statistical significance was assessed by the Student’s t-test; “***” indicates P-value < 0.0005; “**” indicates P-value ˂ 0.005; “*” indicates P-value ˂ 0.05. (F) Expression/purification of MBP-Kermit produces a mixture of the fusion protein and cleaved MBP (arrows), the latter serving as an internal binding control. In pull-down assays, MBP-fused recombinant Kermit, but not MBP itself, indiscriminately binds to GDP- or GTPγS-loaded Gαo-matrices, but not to control GST-loaded or empty matrices. (G) Immobilized Kermit was able to pull down soluble Gαo and human RGS19, but not CG5036. (H) Kermit and control proteins were immobilized on matrix to pull-down Gαo from head extracts of Drosophila overexpressing Gαo in the eyes (using the GMR-Gal4 driver). (I) Solubilized Fz failed to be precipitated by Kermit or CG5036, but was bound by Rab5.

Figure 2. Kermit appears to act downstream from Gαo but upstream from Vang.

Wild-type (yw) wing hairs display uniform proximal to distal orientation (A). Overexpression of kermit under the MS1096-Gal4 driver control results in strong PCP phenotypes including swirling and cells with multiple hairs (B). The UAS-kermit phenotypes are dramatically enhanced by co-overexpression of Gαo (C), but not by its downregulation (D). These phenotypes are also enhanced upon removal of fz (E). Reduction of Vang suppresses the phenotype if achieved by RNAi-mediated downregulation (F), but not by a mere removal of one gene copy (G). The panels (A-G) represent high-magnification images of the dorsal wing sheet within the region framed in magenta in Figure 1A. (H) Quantification of the multiple hair cells induced by Kermit in different genotypes, presented as on Figure 1E. (I, J) Vang-YFP localization in pupal wings of the wild-type (I) and UAS-kermit genotypes (J). Distal is right, anterior is up.

Figure 3. Kermit activity is differently affected by actin- and microtubule-based motors.

Downregulation of jar (MyoVI) by removal of one gene copy (A) or RNAi (B) strongly suppresses the multiple hair phenotype of overexpressed kermit. In contrast, reduction of dynein or kinesin levels by removing one gene copy of the dynein heavy chain (Dhc64C, C) or kinesin heavy chain (Khc, D) enhances the UAS-kermit phenotypes, as does RNAi-mediated downregulation of Khc (E). The panels (A-E) represent high-magnification images of the dorsal wing sheet within the region framed in magenta in Figure 1A. (F) Quantification of the effects of panels (A-E), presented as on Figure 1E.

Figure 4. The model of interrelationship of Go, Kermit, Vang, and motor proteins in PCP.

Microtubule-base motor proteins dynein and kinesin contribute to the asymmetric distribution of Vang in the apical plane, relocalizing Vang vesicles required for the PCP establishment. In contrast, the actin-based MyoVI motor contributes to remove Vang away from the apical membrane and active PCP pool. Kermit transduces the signaling from Go to promote trafficking of Vang via MyoVI.