Rapid Disruption of Dishevelled Activity Uncovers an Intercellular Role in Maintenance of Prickle in Core Planar Polarity Protein Complexes

摘要

class="head no_bottom_margin" id="sec1title">IntroductionPlanar polarity is the coordination of cell polarity within the plane of a tissue (, ). It is most obviously manifested by the orientation of trichomes and bristles in Drosophila or hair structures in the inner ear and skin of vertebrates (, ). Importantly, disruptions in planar polarity have been linked to congenital birth defects and cancer ().At the molecular level, planar polarity is defined as the asymmetric subcellular distribution of planar polarity proteins. During Drosophila wing development, the six proteins of the “core” planar polarity pathway (“core proteins” hereafter) self-organize along the proximodistal axis into stable asymmetric intercellular complexes (A) of variable stoichiometry (). The transmembrane protein Frizzled (Fz) and the cytoplasmic proteins Dishevelled (Dsh) and Diego (Dgo) co-localize at distal junctions, while the fourpass transmembrane protein Strabismus (Stbm; also known as Van Gogh [Vang]) and the LIM-domain protein Prickle (Pk) co-localize proximally. The atypical cadherin Flamingo (Fmi; also known as Starry Night [Stan]) localizes both proximally and distally, bridging the two halves of the complex (reviewed in ). For intercellular complexes to form and distribute to opposite cell ends, activity of all six core proteins is required to enable feedback interactions that are thought to amplify cellular asymmetry (, ).In Vivo Disruption of Dishevelled in the Drosophila Pupal Wing(A) Graphical representation of cells of the wing epithelium depicting the asymmetric distribution of the “core” planar polarity proteins.(B) Schematic representation of Dsh^TEV disruption by heat shock-induced TEVp-induced cleavage at introduced TEVp cleavage sites.(C and D) Twenty-eight hours APF wing epithelium heterozygous for dsh^TEV and hs-TEVp transgenes in a dsh^V26-null background. (C) In the absence of heat-shock, no hs-TEVp is expressed, and the dsh^TEV transgene rescues the dsh-null phenotype, shown by the asymmetric localization of Dsh^TEV (green, C′) and endogenous Fmi (red, C″). (D) After expression of TEVp by a 2 hr heat shock at 38°C, Dsh^TEV localization is absent from the cell membrane (D′), but Fmi localization is maintained (D″). Scale bar, 5 μm.(E) Dsh-EGFP disruption on the basis of targeting with anti-GFP nanobodies fused to the Tom70 mitochondrial translocation signal. Upon Tom70-HA-vhhGFP production via heat shock, Dsh-EGFP is displaced from cell junctions.(F and G) Twenty-eight hr APF wing epithelium heterozygous for dsh-EGFP and hs-Tom70-HA-vhhGFP in a dsh^V26-null background. (F) In the absence of heat shock, Dsh-EGFP (green, F′) and Fmi (red, F″) are asymmetrically localized, and there is no detection of Tom70-HA-vhhGFP by immunolabelling (blue, F‴). (G) After expression of Tom70-HA-vhhGFP by a 2 hr heat shock at 38°C, Dsh-EGFP disappears from the cell membrane and is seen in punctate cytoplasmic spots (yellow arrows, G′), while asymmetric Fmi labeling is maintained at the cell membrane (G″). Tom70-HA-vhhGFP signal (blue) is strongly detected by immunolabelling and is restricted to the cytoplasmic region of the cells (G‴).Insets in (D′), (F‴) and (G′) are increased intensity regions. See also .